JPH09505284A - Cathepsin D is an amyloidogenic protease in Alzheimer's disease - Google Patents

Abstract

  (57) [Summary] Deposition of neurotoxic β-amyloid peptide is a pathological process that occurs in the brain of Alzheimer's disease patients. Disclosed are methods of treating a patient with a therapeutic compound that functions by inhibiting the formation of β-amyloid from amyloid precursor protein (APP). We have identified the aspartic protease cathepsin D as the protease responsible for the amyloidogenic processing of APP. Disclosed are non-toxic compounds that inhibit both the in vitro activity of human cathepsin D and the release of β-amyloid by human cells. Therefore, such an aspartic protease inhibitor has utility as a therapeutic agent for Alzheimer's disease by inhibiting the pathological accumulation of β-amyloid.

Description

Detailed Description of the Invention                  Cathepsin D in Alzheimer's disease                     Is an amyloidogenic protease                                Background of the Invention   This application is a partial continuation application of PCT / US93 / 10889 filed on November 12, 1993. Is a continuation-in-part application of U.S. Patent Application No. 07 / 995,660 filed December 16, 1992. No. 07 / 880,914 filed May 11, 1992 It is a continuation application. 1. Field of the invention   The present invention uses precursors for Alzheimer's disease (hereinafter "AD") β-amyloid tamper To identify proteolytic enzymes with specificity for processing in the cytoplasm Method; Identification of protease inhibitors specific to β-amyloid protein precursors A method specific to a precursor of β-amyloid protein Inhibitors such as cathepsin D, an aspartic protease, and And β-amyloid tampering with inhibitors of chymotrypsin-like serine protease It relates to a method of controlling the formation of keratin. 2. Description of related applications   The assay of the present invention is used to accelerate the formation of amyloid-related peptides in the brain of AD patients. It can be used to identify the protease that regulates the degree. They are simply such professionals It can be used to isolate thease and used as a therapy for AD. It can also be used to identify potential protease inhibitors. To be described later Amilo Major amyloid form that processes id precursor protein (hereinafter "APP") The aspartic protease cathepsin D was identified as an adult protease. Is the application of the assay. Produces amyloid precursor for APP holoprotein Partial characterization of a second serine protease that can be also provided.   AD is usually characterized by progressive atrophy in the frontal, parietal and occipital cortex. It is a progressive degenerative disease of the brain that is manifested. As clinical manifestations of AD, Sexual memory impairment, loss of language and visual-spatial abilities, and behavioral disorders (Mc Khan et al., 1986, Neurology, 34: 939). Global cognitive impairment affects the cerebral hemisphere It is caused by the degeneration of the existing nerve cells (Price, 1986, Annu. Rev. Neurosci ., 9: 489).   Pathologically, the main striking features of the post-mortem brain of AD patients are (1) entangled neurofibrils Pathological lesions consisting of neuroplasm including accumulation of (2) cerebrovascular amyloid deposits; And (3) neuritic plaque. Cerebrovascular amyloid (Wong et al., 1985, PNAS, 82: 8729) ) And neuritic plaque (Masters et al., 1985, PNAS, 82: 4249) are both “A4” or “β- It contains a unique peptide simply named "amyloid".   β-amyloid is an insoluble, highly aggregated, low relative molecular weight of 4,500 It is a polypeptide and is composed of 39 to 42 amino acids. Some evidence streams are: Supporting the role of β-amyloid in the pathogenesis of AD lesions. For example, β- Amyloid and related fragments are toxic to the PC-12 cell line (Yanker et al., 1989, S. cience, 245: 417); toxic to primary neuronal cultures (Yanker et al., 1 990, Science, 250: 279); and causes neuronal atrophy in the rodent brain. And causes a corresponding amnestic response in rodents (Flood et al., 1991, PNAS). , 88: 3363; Kowall et al., 1991, PNAS, 88: 7247). However, familial Alzheimer's disease (FA D) a holo-amyloid precursor protein (this protein When referring to protein, it is referred to as "APP" hereinafter) The strongest evidence from them. N of the β-amyloid peptide sequence in APP Terminal (Mullan et al., 1992, Nature Genetics, 1: 345) or C-terminal (Goate et al., 199) 1, Nature, 349: 704; Yoshioka et al., 1991, Biochem. Biophys. Res. Comm., 178: 1141; Murrell et al., 1991, Science, 254: 97; and Chartier-Harlin et al., 1991, N. ature, 353: 844). FAD by changing the release rate by endoproteolytic Is suggested to cause [Mullan et al., And Chartier-Harlin et al., Both See also).   Kang et al., 1987, Nature, 325: 733 have larger β-amyloid proteins Described as derived from and part of a precursor protein doing. In order to identify this precursor, we designed it from a known β-amyloid sequence. Full-length coding for the protein using a linked oligonucleotide probe A complementary DNA clone was isolated and sequenced. 695 remaining putative precursors Group, which is now called "APP695" (amyloid precursor protein 695) I have.   Subsequent cloning of the gene encoding the APP protein revealed that the A4 region Revealed to be encoded on two adjacent exons (Lemaire et al., 1989, Nuc leic Acids Res., 17: 517), direct A4 accumulation of alternative spliced mRNA The possibility that it was the result of expression was ruled out. This is because A4 accumulation is A4 pepti in APP. Abnormal proteolysis of APP at both N-terminal and C-terminal sites La It must have occurred.   APP695 is the most abundant form of APP found in the human brain, but the other three Forms of APP714, APP751 and APP770 also exist (Tanzi et al., 1988, Natu re, 351: 528; Ponte et al., 1988, Nature, 331: 525; and Kitaguchi et al., 1988, Na. ture, 331: 530). Differences from a single APP gene on human chromosome 21 Splicing produces isoforms of different lengths (Goldgabe r et al., 1987, Science, 235: 877 and Tanzi et al., 1987, Science, 235: 880).   APP751 and APP770 share 40% homology with bovine pancreatic trypsin inhibitor Contains the 56 amino acid Kunitz inhibitor domain. Both of these APP forms have protease inhibition. Possibly harmful (Kitaguchi et al., 1988, Nature, 311: 530 and Smith et al., 1990, Sci ence, 248: 1126), at least one of these forms is probably the protease NekinI. I (Oltersdorf et al., 1989, Nature, 341: 144; Van Nostrand et al., 1989, Nature, 34 1: 546).   The physiological role of amyloid precursor protein has not yet been confirmed. cell Surface receptors (Kang et al., 1987, Nature, 325: 733); Adhesion molecules (Schubert et al., 1 989, Neuron, 3: 680); growth or trophic factors (Saitoh et al., 1989, Cell, 58). : 615; Araki et al., 1991, Biochem. Biophys. Res. Comm., 181: 265; Milward et al., 1 992, Neuron, 9: 129); wound regulator (Van Nostrand et al., 1990, Science, 248: 7) 45 and Smith et al., 1990, Science, 248: 1126) or in the cytoskeletal system. Is proposed to play a role (Refolo et al., 1991, J. Neuroscience, 11: 3888). I have.   Many studies have been conducted to investigate the role of modified APP expression in AD However, the results are inconsistent (for example, see See: Unterbeck et al., 1990, Review of Biological Research in Aging, W. iley-Liss, Inc., 4: 139).   Determine whether changes in the relative amounts of different APP forms are responsible for amyloid accumulation. Research is also being conducted to investigate. The results of such studies are also contradictory However, in general, in AD, the relative expression level of APP containing the Kunitz region is increased. Support the theory. Therefore, transgenic animals with enhanced expression of APP751 Have been shown to show cortical and hippocampal β-amyloid reactive deposits ( Quon et al., 1991, Nature, 352: 239).   Recent studies have shown that the APP fragment (from the N-terminus of A4 to the C-terminus of full-length APP ( Since it consists of about 100 amino acids, it will be referred to as "C-100 fragment" hereinafter) in vitro ( Dyrks et al., 1988, EMBO J., 7: 949) and transfected cells (Wolf et al., 1990, E). Aggregates in both MBO J., 9: 2079 and Maruyama et al., 1990, Nature, 347: 566). I showed you can. C-100 fragment in transfected P19 cells Overexpression has been shown to cause cytotoxicity (Fuckuchi et al., 1992, Biochem. Biophys. Res. Comm., 182: 165).   Furthermore, a C-terminal fragment containing both the β-amyloid region and the C-terminal region is present in the human brain. (Estus et al., 1992, Science, 255: 726). Studies in ectocell lines show that these fragments are produced by the endosome-lysosomal pathway. (Golde et al., 1992, Science, 255: 728).   In summary, the above report shows that a single protein of APP at the N-terminus of the A4 region. Degradation of the protein is sufficient to initiate the pathophysiology associated with AD It suggests. Recent studies have focused on primary cells and cell lines (AD transfectants). Containing the same N-terminus (1-42 amino acids) as β-amyloid. And maybe full length β -Showing that it secretes a 3-4 kDa peptide which may contain amyloid (Haas et al., 1992, Nature, 349: 322 and Shoji et al., 1992, Science, 258: 126). Such peptides are found in the cerebrospinal fluid (hereinafter "CSF") of AD and non-AD patients. (Seubert et al., 1992, Nature, 359: 325 and Shoji et al., 1992) , Supra).   APP also plays a role in the A4 region in the physiological secretory pathway of the APP extracellular region. Cleaved at the site (Esch et al., 1990, Science, 248: 1122 and Wang et al., 1 991, J. Biol. Chem., 266: 16960). This pathway works in several cell lines and is Cause the destruction of the amyloid-forming region (A4 region) of the precursor. like that Evidence has been obtained that the pathway can also work in the human brain (Palmert et al., 1989. Biochem. Biophys. Res. Comm., 165: 182).   The enzyme responsible for normal non-pathogenic APP processing is called "secretase". It is attached. The C-terminal fragment resulting from the action of secretase is the C-100 fragment (see above It is 17 amino acids smaller than the I will call it.   The final pathological accumulation of A4 depends on the specific activity of the pathological and physiological pathways of APP degradation. It is hypothesized to be sex-regulated.   Thus, to explain the accumulation of β-amyloid in the brain of AD patients, There are several possibilities like:   (1) The activity or level of secretase involved in the destruction of the amyloidogenic region deficiency;   (2) Cell sorting of APP which becomes exposed to pathological pathway proteases Change of;   (3) elevated levels of pathological proteases;   (4) Without it, as fast as amyloid is produced Lack of levels of degrading enzymes that break down amyloid; or   (5) Pathological protein caused by mutation in APP amino acid sequence Increased susceptibility of APP to degradation.   Relatively little is known about the regulation of APP sorting in cells. At least The modification of phosphorylation, which is partly due to the modification of protein kinase C activity, It causes altered APP traffic and eventually leads to changes in APP processing. The hypothesis that this is true is increasing (Buxbaum et al., 1990, Proc. Natl. Acad. Sci. USA, 87: 6003). Therefore, treatments designed to alter the phosphorylation of cells are: It caused both quantitative and qualitative changes in the pattern of the C-terminal fragment of APP.   Amyloidogenic APP processing is initially an endosomal-lysosomal phenomenon Was proposed (Golde et al., 1992, Science, 255: 728; C. Haas et al., 1992, Nature, 357: 500), β-amyloid is a differentially processed secreted AP. Released by cultured cells along with the P form (Suebert et al., 1993, Nature, 361: 260). In the secretory pathway in β-amyloid formation or at the plasma membrane Evidence has recently been provided that is consistent with the involvement of proteases in C. Haas et al., 1992. , Nature, 359: 322; Shoji et al., 1992, Science, 258: 126). Recently, FAD's Swed A transfected cell line expressing APP695 that is associated with ish morphology Β-A at a rate 6-8 times faster than cell lines transfected with type APP. It has been shown to release miroid-like fragments (Citron et al., 1992, Nature, 360: 672; Cai et al., 1993, Science, 259: 514 and 1992, Neuro-science Lett., 144: 4. 2) However, a similar study on the effect of the London (V to I) mutation showed that It has been shown to have no effect on Lloyd release (Cai et al., Supra). In some cases Depends on the cultured cells Released amyloid is the APP precursor Valine 594 (number according to reference 1 It contained an unusual β-amyloid form with an N-terminus beginning with (C.H. aas et al., 1992, Nature, 359: 322; Busciglio et al., Proc. Natl. Acad. Sci. USA, 9 0: 2092), but its importance is still unknown. The effect of inhibitors on these systems Strongly supports the involvement of the favored cell compartments in β-amyloid production by Shoji (Shoji et al., 1992). , Science, 258: 126; Busciglio et al., Proc. Natl. Acad. Sci. USA, 90: 2092 and And Haas et al., 1993, J. Biol. Chem., 268: 3021), and some cysteines Suggests no involvement of serine proteases (Shoji et al., 1992, Scien ce, 258: 126; Busciglio et al., Proc. Natl. Acad. Sci. USA, 90: 2092; Haas et al., 1 993, J. Biol. Chem., 268: 3021).   Recently, Nitsch et al., 1992, Science, 258: 304 have reported that some acetylcholine receptors Receptor activation following transfection of cell lines by type In the process concluded to be caused by changes in kinase activity, APP It was shown to cause an increase in sessing and secretion. This latter study has changed Other than correlating the role of phosphorylation, cholinergic neuronal functions specific to AD Provide an association between the default confusion in and plaque pathology.   Despite the above findings, baluns are not affected by any fundamental changes in cell sorting. In order to be able to design a selective and specific therapeutic drug that can reverse the Insufficient knowledge of APP selection.   β-amyloid must be formed by the direct action of proteases. C-10 Identification of so-called "pathological" brain proteases leading to 0 or β-amyloid formation Is a therapeutic protease inhibitor designed to block amyloid accumulation Is an essential step in the attempt to develop a. Identification of such an enzyme is a brain extraction Exist in things Specific activity of the protease in the presence of another brain protease A special assay for the activity of such proteases that would Needs development of a sey.   Such an assay is then used to detect proteases during protease purification. Use i. Finally, it may be needed for drug screening for therapeutic compounds The assay can be used to determine the potential effects of inhibitors of such enzymes.   Some studies have purified and secreted both secretase and the pathological protease of interest. And characterizing. Early studies have shown that only the expected cleavage site in APP An assay was used featuring a synthetic peptide substrate that only mimicked like that Although the assay is useful for measuring the in vitro activity of purified proteases, They contain proteas, as required to monitor protease purification. Have sufficient specificity to allow detection of one protease in the enzyme mixture It is rare. Therefore, those peptidase assays should have the necessary The peptidase activity quantified by this method fails to provide ase specificity. Successfully perform purification of enzymatically active APP candidates from human brain tissue It was not available. Prior to this disclosure, a reliable proteaser of either process No Zea candidate appeared, and the results of various studies were inconsistent.   For example, numerous available studies have found that pathological proteases originated from (Cataldo et al., 1990, Proc. Natl. Acad. Sci. USA, 87: 3861; Haas et al., 1992, Nature, 357: 500); calcium-dependent cathepsin G-like serine protease Is a metal-dependent cysteine protease (Razzaboni et al., 1992, Brain Res., 589: 2. 07; Abrahams et al., 1991, An. N.Y. Acad. Sci., 640: 161); Calpine I (Sima n et al., 1990, J. Neuroscience, 10: 2400); Multicatalytic Pro Thease (Ishiura et al., 1989, FEBS. Lett., 257: 388); serine protease (N elson et al., 1990, J. Biol. Chem., 265: 3836); thrombin (Igarashi et al., 1992) Biochem. Biophys. Res. Comm., 185: 1000); or zinc metalloprotease (WIPO application, WO92 / 07068 by Athena Neuro-sciences, Inc.) ing.   Metallic peptidases (McDermott et al., 1991, Biochem. Biophys. Res. Comm., 179. : 1148); Acetylcholinesterase-related protease (Small et al., 1991, Bioc hemistry, 30: 10795); cathepsin B (Tagawa et al., Biochem. Biophys. Res. Com m., 177: 377); or a broad subsite-specific plasma membrane-associated protease (Sisod ia, 1992, Proc. Natl. Acad. Sci. USA, 89: 6075; Maruyama et al., 1991, Bio-che. m. Biophys. Res. Comm., 179: 1670). Similar contradictions have arisen in identification efforts.   A review of past and current efforts to identify the nature of APP processing enzymes The lack of specificity of the assay used and cerebral tissue It derives from the complex heterogeneity of proteases associated with.                               Summary of the Invention   The present disclosure discloses a tamper for recombinant APP in combination with immunochemical detection of reaction products. Using a special assay based on protein degradation, several APP processing enzymes Describe the method of identification. The assay of the present invention is for assaying β-amyloid from APP. A human brain protease with the exact specificity and proper localization that plays a role in formation. Identify.   The assay format disclosed herein in connection with the identified proteases is Affords the capacity to process many samples, and good for immunochemical detection methods Give sensitivity. In addition, the assay is simple Is easily adaptable to routine use with lab technology and is consistent and reproducible Bring results. These and other improvements are described below.   One goal of the present invention is to discover drugs that can be used to treat AD patients. Is to provide a way to As described above, the 39-42 amino acid peptide β- Proteolytic cleavage of APP that produces amyloid results in amyloid plaque formation disease This is the first step in the pathophysiological process. Several lines of evidence suggest that β-amyloid production Has pointed out the causative role and amyloid plaque as a neurodegenerative feature in AD brain You. These evidences include the following:   (i) Degeneration neurons and dystrophic neuritis and amyloid plaque material Localization (Reviewed in Price et al., 1989, BioEssays, 10:69);   (ii) Evidence that β-amyloid is toxic to neurons in culture ( Yankner et al., 1990, Science, 250: 270);   (iii) β-amyloid degenerates neurons when tested in an animal model And evidence associated with memory changes (Food et al., 1991, PNAS, 88: 3363; Kowal l et al., 1991, PNAS, 88: 7247); and   (iv) Co-segregation of point mutations in APP with certain forms of hereditary AD (Goate et al., 1991. , Nature, 349: 704; Yoshioka et al., 1991, Biochem. Biophys. Res. Comm., 178: 1 141; Chartier-Harlin et al., 1991, Nature, 353: 844; Murrell et al., 1991, Science. 254: 97; Mullan et al., 1992, Nature Genetics, 1: 345).   Thus, the proteolytic conversion of APP to β-amyloid results in AD pathogenesis. Is an essential step in, and in itself is an important target for therapeutic intervention. Seem. Therefore, the related protea The identification of zeoactivity, as well as the development of suitable in vitro screening assays, has led to Therapeutic treatment that can be used as a treatment for suppressing amyloid plaque formation in a person It is an essential prerequisite for the development of Rotase inhibitors.   The present invention was used to discover inhibitors of proteolytic β-amyloid formation. Two inventions that can be made:   (1) A highly purified protease that decomposes APP or decomposes APP Any of the crude biological extracts containing unidentified proteases And an in vitro assay comprising HoloAPP substrate; and   (2) When used in combination with the in vitro assay system described in (1) above, amyloid or Features from human brain capable of forming precursor amyloidogenic C-terminal APP fragments Identification and purification of certain proteases.   The assay detects the in vitro APP degrading activity that produces C-terminal APP fragments. to enable. When used with a crude biological extract, the assay is a source of detectable activity. To monitor the purification of the causative protease or to characterize the protease. Can be used to characterize.   In addition, either purified protease or crude containing unidentified APP-degrading enzyme activity. When used with any of the biological extracts prepared in APP process with chemical or biological compounds co-incubated in It can be used to measure inhibition of singing activity. The block identified by it Harmful Compounds as Inhibitors for Treatment of Amyloid Plaque Formation In Vivo Characterized by AD Patients Can have uses.   The protease identified according to (2) above includes aspartic acid protea Different from cathepsin D and cathepsin G N-tosyl-L-phenylalanine-chloromethyl ketone ("TPCK") ), Α-2 antiplasmin and chymotrypsin inhibitor II from potato Included are chymotrypsin-like serine proteases that are inhibited. Cathepsin D Identification is especially important. We have a cathepsin D of 10.0 kDa and 5.6 kDa, respectively. It is shown that the C-100-like fragment of Izu and β-amyloid-like fragment can be formed .   This finding was either in vitro assay described in (1) above or described in the present invention. Using one of the simpler, higher throughput peptidase assays as described Any purified or isolated cathepsin D can be used to inhibit its activity. Enable exploration.   Furthermore, the specificity of cathepsin D as well as specific aspartic protease inhibitors Much is known about the design of amyloidogenic proteases Identification of cathepsin D as a marker for specific cathepsin D inhibition using established methods. It enables both development of harmful agents and utilization of established cathepsin D inhibitors.   Cathepsin D is, surprisingly, a turn according to Glu (593) -Val (594) (Kang et al., Supra). Hydrolysis of APP at the peptide bond between It is. The preferential specificity of cathepsin D is usually between hydrophobic residues. This information is It can be further utilized in the design of tepsin D inhibitors.   As described above, the thus-identified inhibitory compounds can be used as a biological compound characteristic of AD patients. It has use as an inhibitor for the treatment of internal amyloid plaque formation.   The APP degrading enzyme identified by the use of the present invention was purified and used for the following purposes. Can be used:   (i) Necessary to further correlate AD brain pathology with localization of proteases New immunochemical reagents; and   (ii) To isolate the corresponding protease cDNA. Then use the cloned cDNA A transgenic animal model of AD can be prepared by Therefore, the effect of protease overexpression can be evaluated.   APP degrading enzyme inhibitors identified using the present invention include, for example, affinity It is also useful as a ligand when purifying APP degrading enzyme by chromatography. is there. The column is indirectly enzyme-blocked if necessary through a hydrocarbon spacer arm. It will be filled with an inert matrix (eg agarose) to which the damaging agent is attached . The composition containing the enzyme is then applied to the column and the enzyme Are trapped while all other proteins pass through the column and are discarded. It is. It then modifies the properties of the enzyme and renders it no longer able to bind to the inhibitor Replaces inhibitors by elution with denaturing buffer at pH The enzyme can be released from the column by using a competitive counter-ligand You. In both cases, the enzyme passed through the column and contained no other proteins. It can be collected in good condition. For further details, see, e.g. Palmer, U See nderstanding Enzymes, 1991, Ellis Horwood, New York, Third Edition. . The disclosure of which is incorporated herein by reference.   The assay, which incorporates a synthetic peptide substrate, provides a highly purified protease preparation. Useful for in vitro enzymic studies of products, but generally containing many proteases Allows selective detection of desired protease activity in crude biological extracts Insufficient specificity for. For example, brain tissue contains a wide variety of peptide Specific brain APP, which is rich in glutinase and degrading enzymes and uses synthetic peptide substrates Explain why efforts to isolate degrading proteases were unsuccessful (See background section of the invention above).   Therefore, in Example 3 below, specific assay conditions were determined by synthetic peptide assays. Some peptidases that are unable to degrade APP to generate a C-terminal fragment under conditions Was identified, and the pattern of APP-degrading proteases was identified as brain peptidase. Indicates that it does not look like the corresponding pattern of.   A more limited approach to this problem is incubating with protease-containing fractions. Holo as a substrate, associated with a method of assessing its specific degradation after basation Use of APP. Thus, the present disclosure provides recombinant A with a brain protease fraction. The enzymatic degradation of PP was determined by immunoblotting using an antibody against the C-terminal region of APP. The method described above, which is monitored by   Our assay method is sufficient to include full-length β-amyloid peptide. Formation of C-terminal fragment from Izu APP (proteolysis at N-terminal of A4 region) The process that requires).   Homogenize human brain tissue (non-AD control or AD), then routine ultracentrifugation Soluble fraction (hereinafter “S”) using the method, pelleted after centrifugation at 15,000 g (hereinafter “P2”) ) And the microsome fraction (hereinafter “M”). Membrane M and P2 fractions Is solubilized with the Triton X-100 formulation. Solubility from the obtained M and P2 fractions Fractionation of S and S fractions separately on a Mono-Q strong anion exchange column And separate different brain proteases.   Using a synthetic peptide that mimics the amino acid sequence near the N-terminus of β-amyloid , Peptidases of individual Mono-Q fractions obtained from purification of M, S and P2 fractions Evaluate activity. Column fractions based on recovery of separated peptidase activity peaks Make a continuous pool of.   Pool of peptide activity was purified from the transfected CHO cell line. Establishing Assay Conditions for Detection of Proteolysis of High Purity Recombinant APP Used for. For either the C-terminal region of APP or the β-amyloid region Immunoblot assay to locate C-terminal APP fragment using directed antibody B is developed. Using this assay, potentially containing full-length β-amyloid 6 latents capable of forming a C-terminal APP fragment of a size large enough to Locally different proteolytic activities are identified. APP minutes in the Mono-Q pool Recovery of deactivity is not sufficient for the peptidase activity distribution established in step 2. No relationship can be seen. Studies on inhibitors show that the activity of degrading APP is serine protease activity. It is shown to contain both sex and aspartic protease activity.   The use of peptidase assays to monitor enzyme purification is abandoned . The expression of baculovirus-directed insect cell lines resulted in high levels of recombinant APP The main source of supply to monitor APP degrading activity during protein purification Allows the use of the APP degradation assay as a method. Main aspartic acid prote The ase activity is identified in the fraction from the Mono-Q purification of the P2 fraction.   Further purification and characterization experiments prove that this enzyme is cathepsin D I do. Cathepsin D hydrolyzes holoAPP to produce a 5.6 kDa β-amyloid-like fragment. It turns out to generate   Using aprotinin-sepharose affinity chromatography, Attempts will be made to isolate the aprotinin sensitive APP degrading activity identified above. AP It is possible to cleave P to generate specific C-terminal fragments of 11, 14 and 18 kDa. Motrypsin-like serine protease activity is partially purified. The fragment is full length It is proved by immunochemical technique that it contains β-amyloid.   Through this procedure, we have a high probability that the amyloidogenic potential of APP We have identified several brain protease activities that play some role in degradation. This specification The identified or unidentified activities described therein can be treated with the APP degradation assay. It can be used to screen for therapeutically beneficial selective protease inhibitors. Can be.   As used herein, an "APP substrate" is isolated or from a biological source. Is derived by purification or a gene encoding APP or its analogs. Full-length APP, whether induced by expression of a loaning gene, any such Fragment of a protein (a fragment obtained by digesting the protein or a part thereof) And a synthetic peptide having an amino acid sequence corresponding to a part of APP. Would mean de.   The APP substrate for the assay of the present invention can be provided as a test reagent in various forms. Can be. It is preferably derived from APP695 or the amino acid sequence of APP695. Another APP isoform protein, at least partially corresponding to the column, (see above). Derivatives or analogs of)) are likewise envisaged for use in the present invention. APP695 Schubert et al., 1989, Proc. Natl. Acad. Sci. USA, 86: 2066. By biochemical isolation or purification from such natural sources; or the protein or By the expression of a recombinant DNA clone encoding its functional part (Knops et al. , 1991, J. Biol. Chem., 266: 7285 and Bhasin et al., 1991, Proc. Natl. Acad. Sci. USA, 88: 10307).   Fragments of the APP protein are associated with relevant proteolytic test sample activity (supra). It will comprise a sequence of amino acids sufficient for recognition and cleavage by. Biological material Isolation of APP from E. coli is commonly performed by conventional techniques, such as chromatography, especially affection. Infinity chromatography Purification by Purified APP or its fragments are monochrome Null or polyclonal antibody, which can then be used to It can be used for affinity purification according to routine procedures. As a result The purified APP material obtained is further processed by chemical or enzymatic digestion (eg Can be separated). Useful fragments are relevant proteolytic test sample activity Identified by screening for the desired susceptibility to (supra) Would.   As mentioned above, the APP substrate is a recombinant that encodes APP or a portion thereof. It can also be prepared by expression of a DNA clone. Cloned APP remains The gene may be natural or synthetic in its own right, and the natural gene is a known amino acid. CDN using a degenerate probe based on the acid sequence (Kang et al., 1987, Nature, 325: 733). It can be obtained from A or a genomic library. Suitable recombinant DNA claw Other techniques for obtaining cloned genes, as well as methods for expressing cloned genes The method will be apparent to those skilled in the art.   In detecting the proteolytic cleavage of the APP substrate in the presence of the test sample, various A convenient method can be applied. Some of the currently preferred methods are described below, However, numerous other methods may be used without departing from the inventive requirements of the present invention. It will be appreciated by those skilled in the art that it can be applied to steps. Generally, an APP group To this end any method capable of detecting the occurrence of a quality proteolytic cleavage Available for Such may be signaled by cleavage, such as a colored species. APP to produce optically reactive products such as elementary or fluorescent dyes It can be provided by a suitable design of the substrate.   Another theoretical approach is, for example, by immunoassay 1 or Includes sensitive detection of multiple cleavage products. Currently, such cleavage products are Preferentially the C-terminal fragment of the APP substrate; however, incubation with the sample Any fragments that appear at the time of application can be the target of detection.   Detection of one or more cleavage products characteristic of pathological proteolytic activity is It can be achieved in a number of ways. One such method is (see example below) (Further illustrated in), a procedure commonly known as Western blot is included. Scripture Typically, gel electrophoresis was performed after incubation of APP with the test sample. The components formed in the reaction mixture are separated. The separated protein components are then Transfer to a solid matrix such as a trocellulose membrane.   Then, the membrane-anchored component was treated with a specific anti-reaction against a fragment characteristic of APP degradation. It is reacted with the body and detected by the addition of an enzyme-labeled secondary antibody conjugate. Combined The conjugate is developed with a chromogenic substrate for enzyme labeling.   Various immunoassay formats that can be processed with currently available test systems The formula can also be applied to the detection of APP fragments. Typically, the APP substrate is And immobilize the intact APP produced (eg onto a solid phase). Of the test sample), or alternatively, the test sample is loaded with the immobilized form of the APP substrate. Cubate. The immobilized APP substrate is then transferred to a portion of the APP substrate (APP An antibody that is cleaved from the substrate or directed against (the part that limits the cleavage site) Proteolytic cleavage is detected by reacting with a reagent.   Antibody reagents comprise whole antibodies or fragments containing the antigen binding site (eg Fa b or Fab ') and is of monoclonal or poly It can be of the clonal type. Detection of antibody reagent bound to the immobilization phase should be without the characteristic proteolytic cleavage. And Conversely, loss of antibody binding to the immobilization phase represents APP cleavage. Antibody reagents Detection of binding generally involves the use of labeled forms of such antibody reagents or labeled This may involve the use of secondary antibodies or anti-antibodies.   Capture or immobilization of APP can be accomplished in various ways. Cleavable fragment Antibodies can be generated that are specific for epitopes not present above. So Used to immobilize antibodies such as and to capture or immobilize intact APP be able to. Alternatively, the ligand or hapten is covalently attached to APP. And then capture APP using the corresponding immobilized receptor or antibody. It can be captured or immobilized. Typical ligands useful for this purpose are: The scepter pair is biotin: avidin. Examples of haptens that are useful for this purpose Is fluorescein and digitoxigenin.   The solid phase on which the APP substrate is immobilized or captured can consist of various materials, For example, microtiter plate wells, test tubes, strips, beads, particles, etc. And so on. Particularly useful solid phases are magnetic or paramagnetic particles. Such grain A child is a chemical compound that can be attached to various compounds by simple chemical reactions. It can be derivatized to contain a functional group. Near the container containing the particles The particles can be removed from the suspension by bringing the magnets closer. Like this The particles can be washed repeatedly without tedious centrifugation and filtration. B. It can provide the basis for completely automating the procedure.   Labels for the primary and secondary antibody reagents are from those known in the art. You can choose. If such a label is fluorescent Or a chemiluminescent label, a radioisotope, and more preferably for this purpose Enzymes are alkaline phosphatase, peroxidase and β-galactosidase. It is Ze. The enzymes are stable under various conditions, have high catalytic turnover rates, And can be detected using a simple chromogenic substrate.   Proteolytic cleavage of APP substrates can separate and then detect APP fragments. It can also be detected by Aroma chromatography techniques. High-pressure liquid in this respect Body chromatography (HPLC) is particularly useful. When applying this technology, fireflies Prepare a photolabeled APP substrate. Reaction after incubation with test sample The mixture was applied to a chromatographic column and the fluorescence against intact APP was applied. Observe the differential mobility of sex fragments.   The present invention also provides a method of treating AD patients, either alone or non-toxic and inactive. Effective amount of aspartic protease inhibitor with a pharmaceutically acceptable excipient Is also directed to a method comprising administering to a patient with AD.   The invention further provides such an agent with a non-toxic, inert pharmaceutically acceptable excipient. It also relates to a pharmaceutical formulation containing a spargate protease inhibitor.   The present invention also includes such pharmaceutical formulations in dosage unit form. This is The formulation may consist of individual parts such as tablets, dragees, capsules, caplets, pills, suppositories and And in the form of ampoules, and its inhibitor content is Corresponds to a fraction or multiple. Dosage units include, for example, 1, 2, 3 or 4 doses or Can contain 1/2, 1/3 or 1/4 of a single dose. The single dose is preferably Given in a single dose and usually represents the total daily dose, 1/2, 1/3 or 1/4 Of the active compound.   Non-toxic and inert pharmaceutically acceptable excipients include all types of solids, semi-solids Or it should be construed as liquid diluents, fillers and formulation aids.   Preferred pharmaceutical formulations include tablets, dragees, capsules, caplets, pills, granules. , Suppositories, solutions, suspensions and emulsions, pastes, ointments, gels, gleemes, roche And fine powders and sprays. Tablets, dragees, capsules, Caplets, pills and granules may be formulated with conventional excipients such as (a) Fillers and fillers such as starch, lactose, sucrose, glucose, mannitol And silicic acid, (b) binders such as carboxymethyl cellulose, alginates, Gelatin and polyvinylpyrrolidone, (c) moisturizers such as glycerol, (d) disintegration Breaking agents such as agar, calcium carbonate and sodium carbonate; (e) dissolution retardants, eg Paraffin, for example, and (f) absorption enhancers, such as quaternary ammonium compounds, (g) Wetting agents such as cetyl alcohol and glycerol monostearate, (h) absorption Sorbents such as kaolin and bentonite, and (i) lubricants such as talc, Calcium stearate, magnesium stearate and solid polyethylene Recall, or a mixture of substances listed under (a)-(i) immediately above. Can be   Opaque tablets, dragees, capsules, caplets, pills and granules if desired Conventional coatings and shells that may include agents can be provided, and That they preferentially release in some part of the intestinal tract in a delayed manner if desired It can also be of a composition that is capable of releasing or releasing only the inhibitor. . Examples of embedding materials that can be used are polymeric substances and waxes.   The inhibitor may also be present in microencapsulated form, if appropriate 1 or It is possible to have more than one excipient as described above.   Suppositories will contain, in addition to the inhibitor, conventional water-soluble or water-insoluble excipients such as polyester. Ethylene glycol, fats such as cocoa butter and higher esters (eg C₁₆fat Acid and C₁₄Containing esters of alcohols), or mixtures of these substances Can be.   Ointments, pastes, creams and gels, in addition to the inhibitors, can be used with conventional excipients, eg Examples include animal and vegetable oils, waxes, paraffins, starches, tragacanths, and Lulose derivative, polyethylene glycol, silicone, bentonite, silicic acid, It may contain talc and zinc oxide, or a mixture of these substances.   Fine powders and sprays include, in addition to the inhibitor, conventional excipients such as lactose, tag Luk, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, May contain a mixture of those substances. The spray is a regular spray Agents such as chlorofluorocarbons may be included.   Solutions and emulsions include, in addition to the inhibitor, conventional excipients such as solvents, solubilizers. And emulsifiers such as water, ethyl alcohol, isopropyl alcohol, carbonate Chill, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol , 1,3-butylene glycol, dimethylformamide, oil, especially cottonseed oil, Peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, Glycerol formal, tetrahydrofurfuryl alcohol, polyethylene Contains fatty acid esters of recall and sorbitan, or mixtures of these substances. Can have.   Solutions and emulsions for parenteral administration may also be in sterile form, which is isotonic with blood. You.   The suspension may contain, in addition to the inhibitor, conventional excipients such as liquid diluents such as water, ethanol. Chill alcohol or propylene glycol, and Suspending agents such as ethoxylated isostearyl alcohol, polyoxyethyleneso Rubitol and sorbitan ester, microcrystalline cellulose, metal aluminum hydroxide Um, bentonite, agar and tragacanth, or a mixture of these substances Can be included.   The dosage forms mentioned include colorants, preservatives and odor-improving additives such as peppermine. And eucalyptus oils, and sweeteners such as saccharin or aspartame Can also be included.   Inhibitors of the present invention comprise about 0.1-99.5% by weight of the total mixture, preferably about 0.5-95% It should be present in the above-mentioned pharmaceutical formulation in a concentration of%.   The pharmaceutical formulation as described above may comprise more than one inhibitor, in which case the pharmaceutical formulation as described above. The total amount of inhibitors in the mixture is about 0.1-99.5% by weight of the total mixture, preferably about 0.5-95% by weight. It is. The formulations according to the invention may comprise further active ingredients in addition to the inhibitors according to the invention. .   The above-mentioned pharmaceutical preparations contain, for example, one or more active compounds in a known manner. Alternatively, it is prepared in a usual manner by mixing with a plurality of excipients.   The above-mentioned preparations are orally, rectally, buccal, parenterally (intravenously, intramuscularly or intradermally Bottom), intravaginally, intraperitoneally or topically (fine powder, ointment or drops) Can be Suitable formulations include injection solutions, oral therapeutic solutions and suspensions, gels, Injectable formulations, emulsions, ointments or drops. Ophthalmic and dermatological preparations, Use silver salts and other salts, ear drops, eye ointments, powders or solutions for topical treatment be able to. Furthermore, gels, powders, fine powders, tablets, sustained release tablets, premixes, concentrated Liquids, granules, pellets, capsules, caplets, aerosols, sprays and It is also possible to use inhalants. The inhibitor is another carrier material, eg plastic (E.g. plastic chains for topical treatment), collagen or bone cement. Quality mixing Can be.   Since the site of action is the brain, the inhibitor must cross the blood-brain barrier. Thus, in some cases, for example by attachment to a lipophilic carrier or a lipophilic substituent , For example, hydrocarbon groups such as long-chain alkyl groups, alkenyl groups such as vinyl groups. With any introduction it may be necessary to increase the lipophilicity of the inhibitor. Lipophilic Such modifications to increase the value are routine and within the skill of those in the art. You. For example, R.B. Silverman, “The Organic Chemistry of Drug Design and D rug Action ”, 1992, Academic Press, San Diego, see pp. 361-364 in particular. (The entire contents of which are incorporated herein by reference). Reaching an increase in lipophilicity It is expected that any conventional method will be established. Examples of suitable lipophilic carriers are described in N. et al. Considered by Boder et al. A proposed reversible redox drug delivery system (this is Silverma n, supra, page 362). N. Boder et al., 1983, Pharmacol. Ther. 19: 337 and N.M. Bodor, 1987, Ann. N.Y. Acad. See also Sci., 507: 289. (The entire contents of which are incorporated herein by reference).   Generally, about 0.5-500 mg / kg, in several doses if appropriate to obtain the desired result. The inhibitor may be administered every 24 hours, preferably in a total amount of about 5-100 mg / kg body weight. It is profitable. The single dose is preferably about 1 to about 80 mg / kg, especially 3 to 30 mg / kg body weight. Contains an inhibitor. However, the doses mentioned above should be removed and Nature and weight, nature of formulation and nature of drug administration, and duration of administration or It may be necessary to do so as a function of spacing. Therefore, in some cases , It may be sufficient to treat with less than the amount of inhibitor mentioned above, In other cases, specialization based on expertise facilitates the amount of the above inhibitors. Can be determined.                                   Definition   The following amino acids should be written with the following three-letter or one-letter symbols somewhere in the specification. Can be.                            Brief description of the drawings   FIGS. 1a-1f show a comparison of control peptidase activity compared to AD human cortical subfractions. A dimensional contour plot is shown.   Subfractions were ion-exchanged (Mono-Q) according to Example 1 for P2, S and M fractions. Prepared by separation. N-dansyl-Ile-Ser-Glu-Val-Lys-Met by Mono-Q fraction The enzymatic cleavage of -Asp-Ala-Glu-Phe-Arg-His-Asp-Asp-Asp-Asp (SEQ ID NO: 1) is Performed as described in Example 3. Each plot shows each Mono-Q fraction (( Relative of each fluorescent product (abscissa) obtained by incubation (ordinate) Indicates the amount. The amount of product is represented vertically by contour lines. As the number of contour lines increases As a result, the amount of the specific product increases. Control S (a), AD S (b), control Mono-Q fractions from M (c), AD M (d), control P2 (e), AD P2 (f) for analysis I did. Roman numerals on the right-hand ordinate of the three AD plots are the same as those described in Example 3. Area, which was assayed according to Example 8, showed significant APP degradation activity. It turned out to include sex.   2a-2f are ion exchange fractions of M, S or P2 fractions derived from AD cortex. Immunoblot fraction of APP695-degrading activity associated with specific Mono-Q pools from detachment Represents analysis.   The pools were adjusted to their peptidase activity content as described in Example 3. It was made based on. The immunoblot assay was performed as described in Example 8. Was. A typical assay for the next pool is shown.   Figure 2a: Activity associated with P2 pool V: APP is present in each of lanes 2-6 Was. C-100 from PMTI 73 (lane 1), P2-V free blank (lane 2) , P2-V (lane 3), P2-V + EDTA (lane 4), P2-V + methanol (Lane 5) and P2 -V + Pepstatin A in methanol (lane 6).   Figure 2b: M pool III related activity: APP was present in lanes 2-7. P C-100 from MTI 73 (lane 1), M-III + cystatin C (lane 2), MI II + aprotinin (lane 3), M-III + captopril (lane 4), M-III + EGTA (lane 5), M-III + N-dansyl-Ile-Ser-Glu-Val-Lys-Met-Asp-Al a-Glu-Phe-Arg-His-Asp-Asp-Asp-Asp (SEQ ID NO: 1) (lane 6), no inhibitor Yes M-III (lane 7), and prestained molecular weight markers (lane 8).   Figure 2c: Activity associated with S Pool I: APP was present in each of lanes 2-6. . S-I + N-dansyl-Ile-Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg-His- Asp-Asp-Asp-Asp (SEQ ID NO: 1) (lane 2), SI + EGTA (lane 3), S -I + captopril (lane 4), SI + aprotinin (lane 5), SI + Cystatin C (lane 6), and C-100 from PMTI 73 (lane 7). Leh 1 contains a prestained molecular weight marker.   Figure 2d: Activity recovered in individual Mono-Q fractions from P2 separation of AD: other Mono-Q fractions 38-43 corresponding to the electrical conductivity region where P2 pool VII is observed in some cases Were individually examined for APP degrading activity. Recombinant APP695 for each fraction Incubation was performed both in the absence (-) or presence (+) of. Fraction numbers are given on FIG. The C-100 reference material used is from PMTI 100 Met. Mr indicates a molecular weight marker.   Figure 2e: C-100 product commanded by either PMTI 73 or PMTI 100 Comparing locations of movement: PMTI 73 (lanes 1 and 3) and PMTI 100 (lanes 2 and 4) The protein product of is shown in comparison to the molecular marker (lane 5).   Figure 2f: Typical time course of product formation: APP + M-III over time. t = 0h (lane 1), 5h (lane 2), 20.5h (lane 3), 44.5h (lane) 4) and 55h (lane 5). Molecular weight marker (lane 6), C- 100 PMTI 73 (lane 7) and M-III free APP (lane 8) are shown.   For each of the above Figures 2a-2f, the direction of movement was from top to bottom. 2a-2 In d and 2f, the solid arrow at the top indicates the position of movement of the holo APP, and the arrow at the bottom The solid arrow indicates the moving position of C-100. In Figure 2d, the open arrow is enzymatic. The transfer position of the putative oligomer of the C-100 fragment generated in FIG. Of all inhibitors The concentrations are listed in Table 4 below.   Figures 3a and 3b show the results from further purification of the P2VII pool by gel filtration. Represents   Figure 3a: Pool P2 pool VII fractions from Mono-Q 10/10 chromatography. , Concentrate to 0.25 ml, and concentrate in 10 mM Tris-HCl buffer pH 7.5 containing 150 mM NaCl. It was applied to a tandem array of two equilibrated Superose 6HR 10/30 columns. Elution is 0.3m The column eluate was monitored at 280 nm with a flow rate of 1 / min. Fraction (0.24 ml ) Was collected and the APP digestion assay was performed using a peptidase activity assay and immunoblot. I put it on both essays. The arrow indicates the chromatogram in which the APP decomposition activity was recovered. Indicates the position of the area. The patterns associated with KM bond cleavage (●) and MD bond cleavage (○). Peptidase activity is also shown. Chromatography was performed at 22 ° C.   Figure 3b: Transfer of APP degrading activity compared to standard proteins of known molecular weight. The apparent Mr of APP degrading protease (listed in Example 8) Calculated.   FIG. 4 is a mouse monoclonal antibody raised against β-amyloid peptide. 3 shows peptide epitope mapping of C286.8A. Ma 50ng of synthetic APP695 (597-638) (β-amyloid 1 ~ 42) coated, blocked, then present in the indicated concentration of competing peptide 100 μl C286. Pre-incubated (60 min at room temperature) in the absence or presence. Incubated with 8A (80 ng IgG). Peptides 1-7 are peptides of SEQ ID NO: 1 Note that it refers to Chid. After incubation for 60 minutes at room temperature, wash the plate , According to standard procedures (Wunderlich et al., 1992, J. of Immunol. Methods, 147: 1) Color development using goat anti-mouse polyclonal antibody conjugated with horseradish peroxidase The amount of bound antibody was measured by. The absorbance data at 450 nm is calculated using the following formula: The competitive rate (% C) of antibody binding from the plate to the plate was calculated: β-amyloid 1-42, 1-28, 1-16 and N-dansyl-ISEVKMDAEFRHDDDD (1-7 Inhibits C286.8A binding in a dose-dependent manner, while 12-28, 25-35 and A PP645-695 did not inhibit. Therefore, the reactive epitope for C286.8A is A It is located at the N-terminal 7 amino acids of the 4 region (APP597-603).   Figure 5: APP69 recovered in the ion exchange fraction from the purification of human brain P2 subfractions. 5 Processing activity. A total of 123 fractions were collected from the column. The first 32 fractions are loads And the washing step. The salt gradient started with 33 fractions. Fractions 3-18 (panel a), 21-32 (panel b), 33-38 (panel c) and 39-44 (panel d) images Is shown. For each fraction, in the absence (-) and presence (+) of APP695 substrate Incubation was performed in both. Applicable to APP 695 for 0 and 24 hours as appropriate Incubation is shown. incubation Was performed as follows: Baculo-derived holo APP695 (80 nM) was added to 100 mM Mes buffer pH. 6.5, 0.008% (v / v) Triton X-100, 160 mM NaCl, 6.7 mM Tris (from APP stock solution) Was incubated with 5 μl of each column fraction in a total volume of 15 μl. After 24 hours, the reaction was terminated by the addition of SDS-PAGE sample buffer. Described in Example 8 Immunoblot was performed using the C-terminal polyclonal antiserum of Example 6 as described above. Expanded. The arrow indicates the position of the generated fragment. Fractions 45-86 were also tested, but relatively small It showed no significant activity (hence not shown). Peaks A and B are the main active sites Indicates the location.   FIG. 6 shows the result of purification of P2-derived APP degrading activity by gel filtration: cathepsi Relationship with the elution of protein D. Panel (a) shows P2 peak B on Superose 6HR column. 280 nm elution curve for purification is shown. Panels (b) and (c) are described in Example 5. Of the corresponding APP-C-terminal fragment in the eluted fractions 49-60, measured essentially as Showing processing activity. The arrow indicates the position of the main product band. Panels (d) and (e) Is a rabbit polyclonal antibody against cathepsin D (1/300 dilution). The immunoblot analysis of a fraction is shown. Arrows indicate the location of migration of immunoreactive bands You. Human liver cathepsin D was also analyzed for comparison.   Figure 7: Protease inhibitor specific for protease activity isolated from P2 subfractions. Represents sex. The reaction (32 μl) was started at 37 ° C by the addition of APP, and Achieved: P2 enzyme (2.54 μg / ml) from the 15-25 kDa region of gel filtration (FIG. 6) ) Fraction; APP (168 nM) in 96 mM Mes buffer pH 6.5. After 26 hours, 15 μl of 3 × sample Reaction was terminated by the addition of a dye buffer and the rabbit plasmid for the C-terminus of APP was Subjected to immunoblot analysis with 1/1000 dilution of reclonal antiserum. next The effect of addition of inhibitor is shown: no inhibitor (lanes 7 and 20); 5); 400 μM in ethanol PMSF (lane 9); ethanol only (lane 11); 100 μM E-64 (lane 13); 1 0 μg / ml aprotinin (lane 22); 100 μM pepstatin A in DMSO (lane 26) DMSO only (lane 24). 0 hours (lanes 2 and 30) and 26 hours (lanes 3 and 28) The effect of the incubation of APP) is also shown. Lanes 4, 8, 12, 19, 23 And 27 contained the C-100 standard. Prestained molecular weight markers are lanes 1 and 2 Present in 9. The positions of the 18 and 28 kDa C-terminal generated fragments are indicated by arrows.   FIG. 8 shows cathepses monitored using an antibody against the C-terminal region of APP. 4 shows the time course of APP cleavage catalyzed by SynD. Panel (a) is 86 μM pep Catalysis in the absence (lanes 10-14) or presence (lanes 4-8) of statin A The time course of APP proteolysis by Pusin D is shown. The numbers are after the start of the reaction Indicates the time (hr). The reaction was started at 37 ° C by the addition of APP, and the next initial component concentration Was achieved: APP (82 nM), cathepsin D (9.2 μg / in 89 mM Mes buffer pH 6.5). ml). For samples without pepstatin, add an equal volume of solvent (1.3% v / v methanol). Added. Take aliquots (15 μl) at t = 0, 43, 84, 140 and 215 minutes to give 7.5 Rabbits mixed with μl of SDS-PAGE sample buffer and directed against the C-terminal region of APP Subjected to immunoblot analysis with antiserum. 18 and 28 kDa reaction product positions The position is indicated by an arrow.   FIG. 9: C-terminal processing of APP by P2-derived enzyme or cathepsin D The pH and ionic strength dependence of Panel (a) shows cathepsin D and P2 fermentation Was observed using a sample (peak B in FIG. 5 after gel filtration chromatography in FIG. 6). It also exhibits pH dependence. Panel (b) shows the ionic strength dependence for both enzymes at pH 6.5. Existence. The reaction was started at 37 ° C by the addition of enzyme to achieve the following initial component concentrations: Was : Cathepsin D (9.2 μg / ml) or P2 enzyme from FIG. 6 (11.7 μg / ml), acetic acid Sodium, 100 mM each of Mes and Tris-HCl, and purified APP (79 nM). t = Aliquots (15 μl) are taken at 0 and 3 hours and mixed with 7.5 μl of 3x sample buffer. And immunoblot with C-terminal polyclonal antiserum according to Example 8 Analysis. The positions of the 28, 18 and 14 kDa reaction products are indicated by arrows.   FIG. 10 shows cathepsin D and P2 enzymes (PiE) after further purification on Superose 6HR. N-dansyl-Ile-Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe according to Coke B, FIG. 5). -Arg-NH₂3 shows the cleavage of (SEQ ID NO: 7). Reactions at 37 ° C by addition of enzyme / inhibitor Started, thereby achieving the following t = 0 component concentrations: acetate, Mes and Tris pH Cathepsin D (2.8 μg / ml) in a mixed buffer containing 130 mM each of 5.0 or P2 enzyme from FIG. 6 (17.5 μg / ml), N-dansyl peptide (58 μM), capto Prill (0.3 mM). The sample was pepstatin A (213% in methanol at a final concentration of 3% v / v). μM) or an equivalent final concentration of methanol alone. 0, 2, The reaction was terminated by the addition of 12% (v / v) TFA (10 μl) at 4, 8 and 24 hours. And subjected to HPLC analysis according to Examples 2 and 3. A fee for Table traces are shown: P2 enzyme, t = 0 hours (panel A); P2 enzyme, t = 24. Time (panel B); P2 enzyme + pepstatin A, t = 24 hours (panel C): Cat. Pusin D, t = 0 hours (panel D); Cathepsin D, t = 24 hours (panel E); And cathepsin D + pepstatin A, t = 24 hours (panel F).   Figure 11 shows N-dansyl-Ile- by cathepsin D and P2 enzymes (peak B). Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg-NH₂(SEQ ID NO: 7) pH dependence of cleavage Shows sex. The reaction conditions are essentially as shown in Figure 10. As described. However, adjust the mixed buffer to the indicated pH, and Incubation time is 23 hours for P2 enzyme and cathepsin D It was 5 hours. (a) Cathepsin D cleavage, and (b) P2 enzyme cleavage. Rate of cleavage at the -Glu-Val- (●) and -Met-Asp- (○) bonds in each case Is shown.   Figure 12 is an SDS-PAGE of reaction products from the preparative digestion of APP with cathepsin D. Analysis. Panel (a) is a Coomassie blue stained electric block before band excision. Panel (b) is the corresponding blot after band excision, and Panel (c) is the corresponding series of reactions parallel to those shown in panels (a) and (b). Munoblot analysis (1/100 dilution of monoclonal 286.8A). The reaction of (a) is Starting at 37 ° C with the addition of substrate, the following initial component concentrations were achieved: APP (15.6 μM), cathepsin in 40 mM sodium acetate pH 5.0 containing 30 mM NaCl D (0.17 μM, 7.145 μg / ml). At t = 16 hours, the reaction mixture was placed on a Speed Vac Concentrated to 15 μl, mixed with 7.5 μl of 3 × sample buffer and subjected to SDS-PAGE . The reaction of (c) was carried out in essentially the same manner except that the APP concentration was 3.2 μM. Reduced. In both experiments ((a) and (c)), the incubation was a complete incubation. Lane (lane 3) and in the absence of cathepsin D (lane 4, test). Incubation was performed immediately after addition of the sample buffer) Was. Lane 5 in each case contained a cathepsin D only control, while lane 6 was migrating. Purified APP was included as a standard. Prestained molecular weight markers in lane 1 Exists. In (c), the position of the major immunoreactive product is indicated by an arrow.   Figure 13: Monitored using a monoclonal antibody against the N-terminus of β-amyloid APP components catalyzed by cathepsin D The change over time of the solution is shown. The reaction was started at 37 ° C by the addition of APP and Achieved: APP (448 nM), cathepsic in 83 mM sodium acetate buffer pH 5.0 D (30 nM), and pepstatin A (97.2 μM) when included. When pointed out In, take an aliquot (20 μl) and mix with 10 μl of 3x SDS-PAGE sample buffer. Immunoblot analysis using the combined and monoclonal antibody 286.8A (1/100) I went to The reaction was performed in the absence (-) of pepstatin A (supplied in methanol) or Was performed in the presence (+). All samples received 2.7% (v / v) methanol. Leh Samples 1 and 12 contained prestained Mr markers. Lanes 10 and 18 are C-100Mr markers Was included. Lanes 11 and 13 are 0 and 21:00 respectively in the absence of cathepsin D. The APP was incubated for a period of time. The position of the main product fragment is indicated by an arrow.   FIG. 14 shows the results of synthetic peptides produced by the enzymes derived from cathepsin D and P2 (peak B). The influence of amino acid substitution on the time course of hydrolysis is shown. Reaction by addition of substrate Was initiated at 37 ° C and the following initial component concentrations were achieved: Tris, Mes and acetate buffer p Cathepsin D (2.5 μg / ml) or P2 peak B fermentation in 135 mM buffer of each of H5.0 Elemental (7.5 μg / ml); N-dansyl peptide (58 μM), captopril (0.3 mM), Pepstatin A (213 μM) with or without sodium chloride (75 mM). various Aliquots (30 μl) were taken at different times and adjusted to 12.5% (v / v) in TFA , And subjected to RP-HPLC analysis. Additions for the following substrate / protease combinations The time course of water splitting is shown:   (a) N-dansyl-Ile-Ser-Glu-Val-Lys-Met-Asp-Ala-Glu with cathepsin D -Phe-Arg-NH₂(SEQ ID NO: 7);   (b) N-dansyl-Ile-Ser-Glu-Val-Asn-Leu with cathepsin D -Asp-Ala-Glu-Phe-Arg-NH₂(SEQ ID NO: 3);   (c) N-dansyl by P2 enzyme (peak B of FIG. 5 after gel filtration of Example 6) -Ile-Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg-NH₂(SEQ ID NO: 7);   (d) N-dansyl-Ile-Ser-Glu-Val-Asn-by P2 enzyme (peak B in FIG. 5) Leu-Asp-Ala-Glu-Phe-Arg-NH₂(SEQ ID NO: 3).   EV bond (□ in panels A and C), MD bond (◆ in panels A and C) Cleavage at or at a cleavage time of 4.4 minutes to generate metabolites (patient Channels B and D) are shown.   Figure 15 shows the purification of the solubilized P2 fraction on aprotinin-Sepharose. Experiment Was carried out according to Example 1. (a) is a typical A280 nm elution curve, and (b ) Is an immunoblot assay for APP processing activity in the eluted fraction ( Rabbit antiserum against the C-terminal region of APP). Arrows are major APP decomposition products Indicates movement of an object. Note the appearance of degradation products in fractions 8-13 from acid elution That.   Figure 16 shows the purification of P2-derived aprotinin-binding protease on Mono-Q. (a) shows the A280 nm elution curve, (b) shows the rabbit anti-C-terminal APP antiserum (1/1 for detection) (000 dilution) shows the activity of the eluted fraction, and (c) shows Mab 286.8A (1. The activity of the elution fraction with 6 mg / ml of pure IgG (1/100 dilution) is shown. Three arrows The marks indicate the migration of 11, 14 and 18 kDa C-terminal APP degradation products.   FIG. 17 shows the pH and pH of APP degradation catalyzed by pooled Y serine protease. And ionic strength dependence. Panel (a) shows pH dependence. Start reaction at 37 ° C And the following t = 0 component concentrations were achieved: acetate, Mes and adjusted to the indicated pH values. And Tris in a mixed buffer containing 32 mM each of pool Y protease (from FIG. 16). Fraction 16 of 5 μl), APP (38 nM). After 2 hours, resuspend by adding 7.5 μl of 3x sample buffer. The reaction of the reaction mixture (16 μl) was terminated. Rabbit po to the C-terminal region of APP Using the reclonal antiserum, immunobnnu essentially as described in Example 8, The lot was developed. Lanes 1 and 12 contained prestained Mr markers. lane 9 contains C-100, lanes 10 and 11 at pH 6.5 in the absence of pool Y, respectively. Included APP incubated for 0 and 3 hours. Panel (b) is ionic strength Degree dependence. The reaction was carried out essentially as described under (a). However, loose The buffer was 95 mM Mes pH 6.5 containing the indicated molarity of sodium chloride. each In the absence (lanes 2-7) or presence of pool Y for sodium chloride concentration APP cleavage in (lanes 9-14) is shown. Lanes 1 and 8 are Mr markers And C-100 standard. Arrows indicate migration of 11, 14 and 18 kDa APP fragments You.   Figure 18 shows the inhibitor selectivity of pool Y protease. Reaction by adding enzyme Starting at 37 ° C., the following initial component concentrations were achieved in a volume of 16 μl: 30 mM Mes buffer Pool Y # 3-5 (14 μg / ml) after purification on a Superdex 75 column in pH 6.5, AP P (35 nM). The reaction was terminated by the addition of 7.5 μl of 3 × sample buffer. Example Immunoblot using a rabbit antiserum against the C-terminal region of APP according to 8. Expanded. Data are presented for the effects of the following inhibitors: Panel (a): methanol 860 μM PMSF in lane (lane 4), 400 μM pepstatin in methanol (lane 4) 6) 5 mM benzamidine (lane 8), 350 μM E-64 (lane 9), 7.7 mM EDT A (lane 10), 15 μM aprotinin (lane 11), and 0.1% (w / v) deoxyco Phosphate (lane 15). The following controls were also run: no inhibitor (lane 12), ethano. Pool (lane 3), methanol (lane 5), pool Y only (lane 2), 0 hour APP only (lane 13) and 4 hour APP Only (lane 14). Lanes 1 and 7 are pre-stained Mr marker and C-100 standard, respectively. Indicates a substance. Panel (b): 1.8 μM α-1-antichymotrypsin (lane 2), 156 μM TLCK (lane 3), 46 μM chymotrypsin inhibitor I (lane 4), 119 μM key Motrypsin inhibitor II (lane 5), 4 μM α-2-antiplasmin (lane 6) , 51 μM α-1-antitrypsin (lane 7), 98 μM chymos administered in DMSO Tatin (lane 10), 153 μM methanolic TPCK (lane 12). Inhibition as a control No agent (lane 8), DMSO (lane 11) and methanol (lane 13) were also included . Prestained Mr marker and C-100 standards were applied to lanes 1 and 9, respectively. . Arrows indicate migration of 11, 14 and 18 kDa APP degradation products.   Figure 19 shows that both (1) DMSO and DMSO + 10 μM pepstatin A were used to grow HEK293 cells. No effect (Panel A), (2) DMSO + Pepstatin A treated late pair Conditioned medium from several-phase cultures is significantly lower than cultures treated with DMSO alone FIG. 3 shows that it shows high levels of the 15 KDa C-terminal APP fragment (panel B). Panel A . HEK 293 cells (ATCC CRL 1573, suitable for suspension culture) were mixed with 400 ml of MoAb medium (JRH  Biosciences, Lenexa, Kansas) + 0.2% (v / v) fetal bovine serum in a roller bottle Seeded (final concentration 1 x 10^FiveCells / ml). Additional rotary bottles can be 0.01% (v / v) DMSO (□). Or 0.01% DMSO + 10 μM pepstatin A (◆) was included. Cell culture is 5% CO₂/ 95 % In air at 37 ° C. The number of visible cells in the sample treated with trypan blue There was a constant rate (60%) with respect to time when measured using a hemocytometer. versus At the end of several phases (indicated by the arrow on day 7), conditioned medium was harvested and Beckman Gs -6 tabletop centrifuge at 1500 RPM and immobilized anti-β-amyloid Noclonal antibody (C286.8A) Chromatography on the column. Panel B. Rabbit anti-APP-C terminal Immunoblot with antiserum. Lane 1 is a prestained molecular weight marker Lanes 2-7 are each of fractions 1-6 obtained from purification of medium from DMSO-treated cells. Lanes 9-14 show purification of medium from DMSO / Pepstatin A treated cells. Analysis of each of Fractions 1 to 6 obtained from Lane 8 is C-100 standard (implemented Example 5, from PMTI 100), lane 15 was purified according to method 2 of Example 7. Includes recombinant holo APP695. Fractions 5 and 6 from the treatment with DMSO only Include a missing 15kDa band in the corresponding fraction from Putatin A treatment Pay attention to. Further details are given in Example 12 herein.   Figure 20: Human aspartate protease with APP processing activity Summarize the purification from the brain. a) Solubilized P2 fraction on Mono-Q HR 10/10 column ( Elution curve for purification of protein 140 mg). For each collected fraction, protein Concentration (○), conductivity (dotted line) and absence of 10 μM pepstatin A (●) Is N-dansyl-ISEVKMDAEFR-NH in the presence (△)₂From N-Dancil-ISE The rate of formation is indicated. EV cleavage activity is completely inhibited by 10 μM pepstatin A Was done. Activity is expressed as the% of substrate converted to product by each fraction. b) The APP695 hydrolysis activity is the same as that of a) EV-cleavable peptidase activity. Elute in the degree range. The arrow indicates the position of the fragment of the indicated size (kDa). APP 695 Cleavage was carried out using the following fractions: Fractions 5 (lane 2), 11 (lane 3), 15 (lane 2). 4), 21-26 (lanes 5-10), 30-36 (lanes 11-17). 0 hours (lane 18) or 24 hours (lane 19) incubation with APP695 only or One incubation for each column fraction (not shown) without APP695 Does not produce any fragments Was. Cleavage with purified CD (2 μg / ml) was also performed (lane 1). 0.95 μg An aliquot of incubation mixture containing APP695 was applied to each lane . The pooled fractions for the next study are shown in a).   APP processing: 95 μg / ml of homogeneous Holo APP695 (in Example 7, Method 2) Therefore, the preparation) was added to 40 mM each of acetate, Mes and Tris pH 4.0, 0.002% (v / v) Trito In a total volume of 15 μl containing n X-100 and 30 mM exogenous NaCl, 10 μl of each color Incubate with the water fraction. Relax SDS-PAGE sample to 1x final concentration after 24 hours The reaction was terminated by the addition of buffer solution, and 10-20% acrylamide gradient Tricine gel (N ovex) at a constant pressure of 100 V and then on a Problott membrane (Applied Biosystems). ) Electroblotted on top. Appropriate two bound to alkaline phosphatase Monoclonal to a final concentration of 50 μg / ml by standard sandwich method with secondary antibody Blots were detected using the internal antibody C286.8A.   Peptidase: Add an aliquot (20 μl) of the column fraction to 10 μl of the reaction mixture. To achieve the following initial component concentrations: a mixture comprising 50 mM sodium acetate pH 5.0. Synthetic N-dansyl-ISEVKMDAEFR-NH in combined buffer₂(58 μM), captopril (0 .2mM), methanol (0.3% v / v) and pepstati in methanol when included. A (10 μM). 24 hours later, the reaction was performed by adding 10 μl of 40 μM pepstatin A. Finished. The enzyme product was detected and quantified by RP-HPLC (Example 2).   FIG. 21 shows that immobilized antibody against human cathepsin D degrades human brain APP from solution. It shows that the activity is selectively removed. a) Control (solid line) and anti-cathepsin D ( Dotted line) A 280 nm elution curve for the column. The numbered arrows above indicate the following Point: 1, start of load; 2, flat Wash with equilibration buffer; 3, Elute with 100 mM glycine pH 2.2; 4, 50 mM dietanol Elution with Luamine Hydrochloride pH 11; 5, 100 mM Glycine, 0.5% (v / v) Triton X-1 Elution with 00. b) Selected interstitial fractions from anti-CD (+) or control (-) (1-27), column application (Q pool), purified cathepsin D APP processing activity is shown only for (CD) or APP. Fraction After 40, no APP hydrolysis activity was detected. c) 21-fold concentrated flow-through image Fraction or pooled glycine / Triton eluate (fractions 80-89, 8x concentrated) Immunoblot reactivity against polyclonal antibody against Psin D. Concentrate 10 Performed by precipitation with% (v / v) TCA.   Chromatography: Control and anti-cathepsin D rabbit antiserum as described Rini avid AL chromatography (T.T.Ngo et al., 1992, Chromatography, 597: 1) 01) and by standard methods (R. Axen et al., 1967, Nature, 214: 1302) It was bound to CNBr-activated Sepharose 4B (Pharmacia). APP processing Equivalent from Figure 21b with activity (110 mM sodium bicarbonate pH8 with 100 mM NaCl .1 44 ml of protein in 44 ml) was pooled with immobilized anti-cathepsin DIg Parallel to the same size column (4.2 ml resin) either G or immobilized control IgG And applied. Each of the columns was loaded with 28 ml of 100 mM NaHCO 3 containing 500 mM NaCl._Three p H8.3, 28 ml 100 mM glycine pH 2.5, 40 ml 50 mM diethanolamine hydrochloride pH 11, Finally, 30 ml of 0.5% (v / v) Triton X-100 containing 100 mM glycine pH 2.5 was added to the suspension. It was washed continuously. Fractions collected in acid or base were neutralized with Tris. 0.5m Chromatography was performed at a flow rate of 1 / min and 2 ml fractions were collected throughout. All operations were performed at 4 ° C.   APP processing: Holo APP 695 (final concentration 31 μg / ml) to pH 4.0 Adjusted sodium acetate, Mes and Tris 40 mM, 40 mM NaCl and 0.002 respectively % (V / v) exogenous Triton X-100 in a total volume of 15 μl, 10 μl of each color Incubate with the water fraction. By adding SDS-PAGE sample buffer after 24 hours The reaction was terminated and immunoblotted with monoclonal antibody 286.8A. Analysis. The arrow indicates the position of the generated fragment. High-purity human cathepsin D is an enzyme It was present in the incubation solution at a final concentration of 1.27 μg / ml.   FIG. 22. Immobilized antibody against cathepsin D degrades APP mimicking peptide. It shows that it adsorbs human brain peptidase. a) and b) are respectively N-da Nsil-ISEVKMDAEFR-NH₂Flow-through fraction and pooled glycy for degradation of / Triton eluent pool (# 80-89, Figure 21a, 21-fold concentrated) peptidase activity It is. c) and d) are N-dansyl-ISEVNLDAEFR-NH₂About hydrolysis The corresponding data. In a), the EV hydrolysis activity (○) of the flow-through fraction is shown. It is. In c) LD hydrolysis is shown. Activity bound control (filled Symbol) or anti-cathepsin DIgG (open symbol). It was drawn from the different fractions. The triangle remains at the end of the incubation 10 shows the effect of 10 μM pepstatin A on the amount of uncompetited substrate present. b) and d) in the absence (white bar) or presence (painted) of pepstatin A. The activity was measured with a crushed bar graph).   Method: Addition of column fraction (20 μl) starts the reaction at 37 ° C, Concentration achieved: Acetate, Mes and Tris pH 5.0 at final concentration of 40 mM each Peptide buffer (58 μM), captopril (0.28 mM), meta Knoll (0.1% v / v). When included, pepstatin was at a final concentration of 10 μM. 20 hours later, final concentration 10 The reaction is terminated by the addition of μM pepstatin and according to Example 2 C-10 , RP-HPLC analysis.   FIG. 23 shows cathepsin D from the initial alignment reported in Table 5 to date. Summary of Sequence Alignment of β-Amyloid Fragments Formed from Hydrolysis of APP695 Show. a) βAPP-derived fragment related to C286.8A immunoreactivity on immunoblot N-terminal sequence (lane for immunoblot taken from Figure 12c: lane 1, βAP P695 only; lane 2, βAPP695 + CD). The arrow indicates the immunoblot band , A sequencing block with sufficient sequence and size to contain the C286.8A epitope. Associated with the fragment identified in the corresponding segment of G. Uncertain in lower case Means a perfect sequence alignment. The amino acid numbering of APP695 is Kang et al., 1987, Nature. , 325: 733. b) Contains the C286.8A epitope (hatched) or contains ΒAPP695 fragmentation pattern including all recovered fragments that were not (white). Each piece The length is calculated from the indicated fragment size (from SDS-PAGE) and the assumed average residue mass of 110. It is drawn in proportion to the estimated number of residues per fragment calculated. For a certain fragment size It does not take into account the potential effects of glycosylation. Β in mature βAPP695 The position of AP (segments with diagonal lines) is also shown. In b), the hydrolyzed bond Is βAPP₆₉₅Residues 122-123 (F-V); 303-304 (Y-L); 405-406 (L-Q); 459-4 Corresponds to 60 (L-R); 532-533 (L-P); 549-550 (F-G); 593-594 (E-V).   Method: Reaction was performed as described in Figure 12. Specific to complete incubation CD-dependent immunoreactivity in immunoblot containing specific fragments or in parallel. A segment of a Coomassie blue-stained electroblot that was co-migrated with the probe. Then in the vapor phase Sequencing was performed on an Applied Biosystems 477 type protein sequencer. βA 80% of PP was hydrolyzed into small fragments. Detectable N-terminal sequence 12.7 picomoles Was applied to the gel in an amount equal to   FIG. 24 shows the effect of ΔNL mutation on the hydrolysis of βAPP695 by cathepsin D. The result is shown. The reaction was started at 37 ° C to give the following component concentrations: 0.128% Triton X- High-purity human cells in 100 mM each containing 40 mM sodium acetate, Mes and Tris, pH 5.0 Tepsin D (10 μg / ml), purified βAPP695 or βAPP695ΔNL (84 μg / ml) , Expressed according to method 2 of Example 4 and purified according to Example 7). Take an aliquot (20 μl) and follow the C286.8A module according to the Materials and Methods section. At -80 ° C until tested for immunoblot reactivity against monoclonal antibody It was stored frozen. Shaped at 0 (lane 1), 5 (lane 2) and 20 (lane 3) hours The immunoreactive βAPP695 hydrolysis product produced is shown. 20:00 without CD In-between βAPP695 (lane 4) or βAPP695ΔNL (lane 9) Bastion is shown. Lane 5 contains recombinant C-100 (βAPP596-695, Example (Expressed using PMTI-100 according to 5) is shown. Arrows are 10-12kDa And migration of the 5.5 kDa C-terminal APP fragment.                            Detailed description of the invention   The present invention will be more specifically described with reference to the following non-limiting examples.Example 1. Isolation of human brain protease   Two general approaches were taken for protease isolation. In the first study below Brain protector as described in "Method 1" Zea activity was isolated. Characteristics of enzyme activity obtained from ion-exchange chromatography. The signature is described in Examples 3 and 8, which degrades APP from recombinant CHO cells. (Example 8) was relatively inactive as a peptidase (actually, Example 3) resulted in the identification of 6 different activities.   One of the six activities was subsequently identified as Cathepsin D (Example 9), It was further characterized according to gel filtration chromatography.   Several human brain serine proteases described in Example 8 (Table 4) were Another approach that attempts to purify tea was to implement "Method 2." This one The method uses an aprotinin-sepharose-affected serine protease affi Serineps based on nity purification and also showing C-terminal processing ability of APP This led to the identification of the Rotase (Example 10).Method 1:   (i) Subdivision of cells. Frontal cortical polar field 8 dissection from four different age AD patients Weigh one piece (4.5g) while freezing (-70 ℃) and add to 150ml of 0.32M sucrose at 4 ℃. Then, cut it into small pieces with scissors. Divide this suspension into 100 ml of Elvehjem glass − Homogenized with a Teflon stick (10 round trips). The combined homogenate is So It was centrifuged in a rval SS-34 rotor (1000 g x 10 minutes).   Remove the soft pellet, rehomogenize and centrifuge as above. Was. Combine the supernatants from each extraction and centrifuge for 30 minutes at 15,000 g in a Sorval SS-34 rotor. Heart isolated. The resulting "P2" pellet was vortexed to add 100 ml of ice-cold 0.32M sieve. It was resuspended in sucrose and stored frozen at -70 ° C. Supernatant from the last centrifugation at 105,000 g Centrifuge for 60 minutes to give a supernatant or soluble fraction (“S”) and microsome fraction (“M”) ) Was resuspended in 60 ml 0.32M sucrose. Both S and M were stored frozen at -70 ° C.   (ii) Solubilization. Membrane control or AD subfraction (P2 or M) adjusted to the following conditions Solubilized by: 50 mM Tris-HCl buffer containing 2% (v / v) Triton X-100 The pH of the buffer solution was 7.5. After stirring for 3.5 hours at 4 ° C, the suspension was stored in a Beckman 70 Ti rotor at 105 It was centrifuged at 1,000 g for 60 minutes. The following final protein concentrations were used for solubilization: (3 for P2 .9-4.0 mg / ml); for M (1.4-1.6 mg / ml). The solubilized supernatant should be used next time. At -70 ° C. Soluble fraction was not treated with detergent and added with 1M stock solution. The pH was adjusted to 50 mM Tris-HCl, pH 7.5 by addition.   (iii) Ion exchange chromatography. Chromatography is 50 ml Rheodyn e Stainless Steel Loop Injector Type 7125 is installed and Mono-Q HR 10 / Gilson gradient liquid chromatography connected to 10 columns (Pharmacia, Piscataway, NJ). This was done using a mattograph machine (type 305 and 306 pumps). Absorbance of column effluent Degree at 280 nm using a Pharmacia UV-M detector and Kippen-zonen chart recorder. Monitored.   P2, microsome (M) or soluble (S) protein fractions are loaded onto the column 2 ml / using 50 mM Tris-HCl, pH 7.5 (electric conductivity 1.8 mU at 4 ℃). Equilibrated at a flow rate of minutes. Then load the column until the A280nm of the eluate drops to zero. Washed with equilibration buffer. The column flow rate was increased to 4 ml / min during this wash.   The protein was eluted as follows:   Solvent: A = 50 mM Tris-HCl pH7.5               B = 50 mM Tris-HCl pH 7.5 containing 1 M NaCl   Program: 0-50% B over 70 minutes               Maintain 50% B for 10 minutes               50-100% B over 10 minutes               Maintain 100% B for 10 minutes               Rebalancing 4 ml fractions were collected throughout the chromatography. Set the next protein load for each Applied for lamb elution: P2, 97 mg (control) and 95 mg (AD); S, 50 mg (control) 68 mg (AD); M, 36 mg (control) and 31 mg (AD).   In the first experiment, the elution fraction was measured at A280nm, total protein (Bradford assay) And peptidase activity (described in Examples 2 and 3) were monitored. Next A pool was prepared based on the peptidase activity in (Example 3), and CHO cell-derived AP was prepared. Those pools were tested for their ability to process P-terminally C (Example 8). Described).   In all subsequent experiments, the elution fraction was induced by baculovirus-directed expression. The recombinant APPs were tested individually for C-terminal processing ability (Example 9). ).   (iv) Gel filtration chromatography. A typical example of gel filtration is depicted in FIG. More generally, chromatography is performed as described below. gave. The Mono-Q fraction containing APP-C terminal processing activity from P2 purification Pool, concentrate to less than 0.25 ml, and add 10 mM Tris-HCl buffer p containing 150 mM NaCl. Applied to a tandem arrangement of two Superose 6HR columns equilibrated with H7.5. 0.3m from beginning to end A flow rate of l / min was used. Fractions were analyzed at A280 nm, total protein (Bradford assay), Monitor for peptidase activity and C-terminal processing activity of recombinant APP Ringed.Method 2:   (i) Subdivision of cells. Performed essentially as described in Method 1 above.   (ii) Solubilization. Performed essentially as described in Method 1 above.   (iii) Aprotinin-Sepharose chromatography. Soluble (23 0 mg), P2 (216 mg) or microsome fraction (47 mg) in advance with 20 mM Tris-HCl Aprotinin-Sepharose column (Sigma, column equilibrated with buffer pH 7.0). It was loaded on the tag number 42268, 1.5 × 10 cm). Once loaded, load the column into equilibration buffer ( 100 ml), then 50 mM sodium acetate buffer containing 500 mM sodium chloride Elution was carried out at pH 5.0 (60 ml). The flow rate was 1.0 ml / min throughout. Elution fraction (4 ml) Was monitored at 280 nm and analyzed using the Bradford protein assay and then Induced by expression in a baculovirus system as described in Example 8 Recombinant APP was used to investigate APP-C terminal processing activity. Active image Anti-APP-C terminal antibody (see Example 6 for antibody production method) Approximately 11, 14 and 18 kDa fragments that can be detected on immunoblots using Can be formed.   (iv) Ion exchange chromatography. Aprotinin-P on Sepharose The active fractions from the purification of 2 fractions were pooled and -Mono-Q buffer dialyzed against HCl pH 7.5 and pre-equilibrated with dialysis buffer. Applied to rum (HR 5/5). Once loaded, essentially as described in Method 1 above. The column was eluted. The active fraction (2 ml) was measured at A280 nm and total protein (Bradford And the ability to process C-terminal processing of APP from baculovirus. I monitored it. A broad peak of APP degrading activity was observed, which is -Monochrome directed against the N-terminus of the C-terminal antibody and β-amyloid peptide React with both null antibodies (see Example 6 for antibody production) 11,14 And an 18 kDa C-terminal APP fragment could be formed.   Based on the A280nm distribution across the area containing the active fraction, each unique A280nm Three active pools were prepared that overlapped with the oak. These pools have the following electrical conductivity Areas included: Pool X (12.2 to 14.4 mmho), Pool Y (14.9 to 18.9 mmho) and Pool Z (20.2-22.9mmho).   (v) Gel filtration chromatography. 2 ml of pools X, Y and Z (pool X and Y) or 3.6 ml (Pool Z) and concentrated with 150 mM NaCl. Superdex 75 column pre-equilibrated with 50 mM Tris-HCl buffer pH 7.5 (Pharm acia, Piscataway, NJ). Once loaded, load the column with equilibration buffer. Eluted. Chromatography was always performed at a flow rate of 1.0 ml / min. Fraction (1 ml ) A280 nm, total protein (Bradford assay) and baculo-expressed APP It was monitored for C-terminal processing activity. Each of the following standard proteins A calibration curve for gel filtration was prepared by chromatography on: thyroglobulin (67 0kDa), bovine γ-globulin (158kDa), ovalbumin (44kDa), myoglobin Bottle (17.5kDa) and vitamin B₁₂(1.35kDa).Embodiment 2. FIG. Development of peptidase assay   At the junction between the "extracellular" region and the N-terminus of the β-amyloid peptide region In human brain tissue that may have suitable specificity for APP hydrolysis of A peptidase assay was developed that allows high throughput detection of lotase. Choice The technique used is related to the subsequent detection of fluorescent peptide products by RP-HPLC separation. Then, a dansylated peptide substrate and post-column fluorescence detection were used.   Evolution of peptide substrate sequences: N-terminus of human APP β-amyloid peptide sequence Fluorescently labeled dodecapeptide substrate containing the same amino acid sequence found near the region Was prepared by solid phase peptide synthesis. Peptides are Fmoc / NMP-HoBt chemistry (Fields et al. , 1990, Int. J. Peptide Protein Res., 35: 161; Knorr et al., 1989, Tetrahedron.  Letters, 30: 1927) on an Applied Biosystems 430A peptide synthesizer. Synthesized. Usually 90% trifluoroacetic acid, 4% thioanisole, 2% ethanedi Cleavage and deprotection of peptides in thiol and 4% liquefied phenol for 2 hours at room temperature Was.   However, the peptide: N-dansyl-Ile-Ser-Glu-Val-Lys-Met-Asp-Ala- Glu-Phe-Arg-His (SEQ ID NO: 2) is desirable when incubated with crude tissue fraction It turned out to be badly digested by carboxypeptidase. Carboxypep The following improved substrates were designed to weaken the digestion of Tidase: N-dansyl- Ile-Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg-NH₂(SEQ ID NO: 7) and N- Dansyl-Ile-Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg-His-Asp-Asp-Asp-Asp (SEQ ID NO: 1).   This latter peptide was digested with carboxypeptidase in the presence of crude tissue fractions. Are relatively insensitive to Peptidase of Example 3 Used for profiling studies. The C-terminal α-amide substrate (SEQ ID NO: 7) is further It was used for the peptidase study of Example 9 using the purified enzyme fraction. Each of them Peptide degradation was monitored using the HPLC protocol of Example 3 below.Embodiment 3 FIG. Measurement of peptidase activity in subfractions of normal control brain and AD brain   (i) Incubation. An aliquot of the column fraction described in Example 1 (20 μl ) Was incubated with 10 μl of the reaction mixture to give the following component concentrations: : N- in a mixed buffer containing 100 mM each of MES, Tris and acetate, pH 6.5 Dansyl-Ile-Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg-His-Asp-Asp-Asp-As p (SEQ ID NO: 1) (50 μM), captopril (300 μM).   Incubation with the ion exchange fraction was carried out at 37 ° C for 24 hours, after which the final concentration was The reaction was terminated by adjusting to 3% (v / v) TFA.   (ii) HPLC quantification of proteolytic products. For HPLC analysis, use a dual solvent supply system, column Use the Hewlet-Packard HP1090 complete with oven and auto-injector It was carried out. HPLC CHEM station (DOS series) and appropriate data analysis Inline Gilson 121 type filter fluorometer (excitation 310-410nm, Fluorescence detection (post-column) was performed using luminescence (480-520 nm).   An aliquot of the above acidic incubation mixture (usually 10 μl) was added to C18 5 Hypersil 5 μm C18 column (100 × 4.6 mm) with μm guard column (20 × 4.6 cm) ). 100 mM sodium acetate buffer containing 27% (v / v) acetonitrile, pH Equal separation was performed using 6.5. Identification and comparison with synthetic peptide product migration , It's structure The structure was confirmed by PTC-amino acid analysis and FAB-MS (see Table 2 below).   The retention times of some of the metabolites listed in Table 2 above are variable in the HPLC setting. N-dansyl-Ile-Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg-His ( Note also that the cleavage in column number 2) differs from that shown in Example 2. For example, the study that provided the data listed in Table 2 included a guard column on the HPLC column and column. It has a relatively long retention time due to the chromatography performed on the ram.   All experiments were performed enzymatically by analysis of synthetic standards in parallel with experimental samples. The HPLC column was calibrated for daily variations in the retention time of the product made. HP C Using the HEM station data collection software, the proteins of individual ion exchange fractions Data were collected on the quality degradation metabolite distribution.   The area under the curve for each of the six cleavage products and their retention times are reported in a peak table file. I saved it. Accumulate all peak tables, and EXCEL (in programming language C Converted to (6 × n) parcel alignment (using the written custom utility). Where n = Mono-Q fraction Is a number. Each row in the alignment represents a single peptidase assay from the Mono-Q fraction. Row Zeros are inserted between each column of data to artificially establish a numerical gradient in the direction. You.   SpyGlass extracts this alignment and determines the Mono-Q fraction, cleavage site and And transform it into a three-dimensional surface whose area% is three axes. Contours are SpyGlass Pro Limited by the following criteria set manually in grams: The first contour is Connect the continuous regions of the plot where 1.5% substrate conversion to the specific product was observed. As well Contours connecting regions of 5%, 10%, 20%, 30%, 40% and 50% substrate conversion Is also displayed continuously. The resulting contour plot has fraction numbers on the ordinate and the The tide bond cleavage site is on the abscissa and the amount of product formed is Represents a brain peptidase map represented.   Analysis results of peptidase distribution in control and AD brain subfractions: FIG. 1 shows a control and a further subfractionation by ion exchange chromatography. Obtained for cleavage of N-dansyl peptide substrate by P2, S and M fractions of AD Figure 3 shows a comparison of the peptidase distributions obtained. This analysis is a subdivision of control and AD brains. Allows the identification of a large number of potentially different peptidase activities throughout the minute.   In each assay (a-f in Figure 1) the cleavage activity at each peptide bond The amount decreased in the following order: V-K> K-M> M-D> S-E> E-V, but K-M and M-D cleavage were the largest. Is also noted. Because they are the part of APP hydrolysis that leads to C-100 formation This is because there is a high possibility that it will represent rank. The metabolite recovered in 1.6 minutes was Probably because treatment with hydrogen hydride yields products with the same retention time (rt) This is probably because methionine in the substrate was oxidized to sulfoxide. 1.0, 1.3 and A metabolite with an rt of 3.3 minutes is currently unidentified.   For K-M cleavage, both the control and AD enzyme spectra and peak activity were noted. The overall level of activity fell in the following order: P2> S = M. This is MD cleavage at AD , But the order for the control fraction was S> P2 = M .   The most abundant peptidase peaks found in AD (connected by multiple contour lines) The following number of clearly resolved peptidase peaks We were able to distinguish: P2, 3 KM peaks and 1 MD peak; M, 3 KM peaks And two M-D peaks; no S, K-M peaks and one M-D peak. A corresponding control and A A quantitative comparison of the levels of the more abundant peaks between the D subfractions reveals only one notable difference. I made it soft. The difference was observed in the microsomal fraction, the control M fraction had 75 fractions. However, in the AD fraction, the same region clearly contains doublet. I was there.   Due to potential mutations in peptidase levels between normal and pathological populations, It does not make much sense to emphasize the quantitative difference in peptidase levels between teratosis and AD Yes.   Generally, there may be quantitative or significant qualitative differences between the control and AD fractions It can't be ignored, but the overall distribution (profile) is more abundant. Very similar to the only obvious qualitative difference that is apparent among the enzyme activity peaks Looked like.   (iii) Integration of peptidase activity into separate pools. N-Dancil-Ile-Ser-Gl u-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg-His-Asp-Asp-Asp-Asp (SEQ ID NO: 1) substrate Based on the peptidase activity distribution obtained using Column fractions from on-exchange separation were combined into a continuous pool (12 pools for M , 13 pools for P2 and 14 pools for S). Control in each case with AD fraction Pool the same area of the chromatographic curve for the fractions Be careful (even if no protease is detected in the corresponding control region) ), And monitor the conductivity of each pool using a YSI 35 type conductivity meter By doing so, the accuracy of this process was confirmed.   Fractions throughout the pool were maintained at 4 ° C and then stored frozen at -70 ° C. Each pep The Tidase pool was concentrated using an Amicon Centriprep-10 membrane. Before concentration, each Cent The riprep membrane was washed in 50 mM Tris / HCl, pH 7.5. Peptida containing a volume of 5 ml or more For the pool, 15 ml Centriprep was used. Du Pont So Concentrated on a rval tabletop centrifuge at 2700 rpm for about 40 minutes. Pool containing less than 4ml is 2m 40 at 5000 rpm on a Du Pont Sorval centrifuge (SS34 rotor) using l Centriprep Concentrated for a minute.   After concentration, each concentration pool was washed with the same volume of 50 mM Tris / HCl, pH 7.5. Centrifugal processing The pool was maintained at 4 ° C for the duration and then stored at -70 ° C. Those pools are electric Listed in Table 3 along with conductivity and final concentration volume. Embodiment 4. FIG. Expression of recombinant APP695   This example describes how to express holoAPP695. Holo APP695 It was then purified as described in Example 7 and then the APP content described in Examples 8-10. Used as a recombination substrate for digestion assays. Two approaches were used for expression.   First, the CHO cell expression system was used to express APP695. As a substrate Experiments with this source of APP showed that Mo from purified P2, S and M fractions of human brain. results in the identification of 6 different proteases that can be detected in a continuous pool of no-Q fractions Initial activity measurements were included. Next, an expression system using the baculovirus command system Recombinant APP695 was obtained. As a result, a larger amount of APP695 is produced, It becomes possible to carry out the detailed studies outlined in Examples 9 and 10.   Two expression methods are described below. Method 1: Chinese hamster ovary expressing holo APP695         Development of (CHO) cell line   (i) Preparation of vector. 2. Known APP695 cDNA from FC-4 (Kang et al., Supra). The 36Kb NruI / SpeI fragment was filled in with the large fragment of E. coli DNA polymerase I. And KS Bluescript M13 + (Stratagene Cloning Systems, La Jolla, CA) Blunt-ended at the SmaI cloning site of pMTI-5 (under the control of the T3 promoter) APP695) was prepared. The following oligos: 5'-CTCTAGAACTAGTGGGTCGACACGATGCTGCCCG Site-directed mutagenesis using GTTTG-3 '(SEQ ID NO: 8) (Kunkel et al., 1987, M ethods in Enzymology, 154: 367) to create new optimal Kozak consensus DNA sequences. Then, PMTI-39 was produced. This plasmid is then replaced with valine at position 614 by glutamine. Change to acid (Numbering of open reading frame follows Kang et al., Supra) Site-specific sudden change PMTI-77 was prepared by modification by induction.   Not the full-length APP cDNA containing the optimal KozaK consensus sequence and the Val to Glu mutation, It was excised from PMTI-77 by partial digestion with I and HindIII. Then this 2.36Kb APP6 The 95 fragment was gel purified and NotI / HindIII cleaved pcNAINeo (Invitrogen  Corp., San Diego, CA), and APP695 expression is a CMV promoter. PMTI-82, which is placed under the control of, was prepared. Sequence analysis of Val to Glu mutations Use this vector for more confirmation and for stable transformation of CHO cells. Was.   (ii) Generation of stable CHO cell line expressing APP695 mutein. APP6 Chinese hamster ovary K-1 cells (A I used TCC CCL 61). Contains APP695 and neomycin drug resistance marker 20 μg of expression plasmid was transferred to Bio-Rad Gene Apparatus (Bio-Rad Laboratories, Ric 1 x 10 in 0.5 ml PBS by electroporation using hmond, CA)⁷ CHO cells were transfected. 1200V with 25μf capacitance A single pulse of was applied to the cells.   After electroporation, the cells are incubated on ice for 10 minutes and then centrifuged. Collected by heart separation. 5x1 cell pellet in Alpha MEM, 10% fetal bovine serum 0^FourResuspend to a concentration of cells / ml and add 1 ml aliquots to 5 24-well tissue culture classes The plate was distributed to each well. After 48 hours incubation, 1 mg / ml Add 1 ml of medium containing Geneticin (GIBCO-BRL, Grand Island, NY) and incubate. Solution and the medium containing the drug is replaced every two weeks, Cells containing the syn drug resistance marker were selected.   Drug resistant cells were tested for APP695 expression by Western blot . Cells that are positive for APP695 expression are diluted by limiting dilution Cloned, isolated individual clones and tested for APP695 expression did. Cells that are positive for APP695 expression are subcultured to produce a large amount of APP695-expressing cells. Grow in roller bottles for production and subsequent isolation of recombinant proteins. Method 2: Recombinant holo APP695 and h using baculovirus-infected insect cells Expression of APP695ΔNL   (i) Preparation of recombinant vector. Baculovirus for expression of wild type APP695 The vector pVL1392 (Invitrogen) was cut with XbaI (in polylinker) and pM Ligated with gel-purified 2.36Kb XbaI fragment from TI-39 (KS Bluescript M Having NruI / SpeI of APP695 in the SmaI site of 13+, T3 orientation, SmaI / NruI With new Kozak and XbaI sites at the blunt fusion site). Created pMTI -103 was transformed into DH5α, selected for Amp and plasmid D A lithium preparation of NA was made for transfection.   To prepare βAPP695ΔNL vector, sense cDNA sequence of βAPP695 Primer (5'-agg aga tct ctg aag tga atc tag atg cag-3 ') (SEQ ID NO: 10 ) And antisense primer (5'-cat gaa gca tcc ccc atc gat tct taa agc-3 ') To amplify by PCR and encode mutations from 595K, 596M to NL A 0.7 KbβAPP cDNA fragment was obtained. This PCR fragment was digested with BglII / ClaI, And insert into the vector containing full-length βAPP695 that had been cleaved with the same enzyme in advance. did. A 2.5 Kb XmaI / SpeI fragment from this vector containing full-length βAPP695ΔNL. Fragment and then XmaI / Xb of the baculovirus expression vector pVL1393 (Invitrogen) It was inserted at the aI site.   (ii) cells and viruses. ATCC (the American Type Culture Collection) Purchased from Spodoptera Frugi Perda (Spodoptera frugiperda) (Sf9) Cells were cultured in TNMFH medium containing 10% fetal bovine serum (Summers, M.D. and Smith, G.E. , 1987, A Manual of Methods for Baculovirus Vectors and Insect Cell Proce dures, Bulletin, No.1555, Texas Agric. Exp. Stn. and Texas A & M Univ., They were grown as suspension cultures at 28 ° C in University Station, TX). Wild type Ac MNPV DNA was purchased from Invitrogen, San Diego, CA.   (iii) DNA transfection and plaque assay. Calcium phosphate Directly with purified plasmid DNA (4 μg) using the method (Summers et al., 1987, supra). Co-transfection of linear viral DNA (1 μg) into Sf9 cells And by homologous recombination at the polyhedrin locus in the AcMNPV genome. Then, the foreign DNA was inserted. The virus released by the transfected cells Ruths were purified by two plaque assays (Summers et al., 1987, supra), where By visually screening for polyhedrin-negative plaques with Recombinant virus was identified. The purified recombinant virus culture was then Screen for their ability to produce APP in infected cells by blot analysis. I learned.   (iv) Recombinant protein production. 1x10 in TMNFH medium containing 10% fetal bovine serum⁶ For a 5 l batch of SF9 cells grown as a suspension culture at a concentration of cells / ml, M.O.I. = 1 was infected with the recombinant virus. Cells were harvested 24 hours after infection and recombinant Cell lysates were prepared for purification of proteins.Embodiment 5 FIG. Production of recombinant C-100 reference material by transient infection of mammalian cells Development of expression vector for production   The C-100 peptide fragment extends from the N-terminus of A4 peptide to the C-terminus of full-length APP. Including the C-terminal part of APP (see "Background" section above). C-100 disconnection The strip is the first degradation product alleged to result in the final formation of the A4 peptide.   In one aspect of the invention, it is a Hela S3 cell expressing recombinant C-100 (ATCC CCL 2.2). These cell lysates were combined with the brain fraction subdivided by Mono-Q chromatography. Immunoblot was performed in parallel with the incubated recombinant APP sample (see Example 3). It was analyzed by the Dot assay. Migration and detection of the C-100 fragment is a pathological proteaser. Not only as a size marker indicating the origin of the product formed by It also serves as a positive control for the immunodetection of specific C-terminal APP fragments.   A comparison of the size of the enzymatically made fragment and the size of the C-100 fragment is made enzymatically. The resulting fragment resulted from cleavage near the N-terminus of the A4 peptide, or Did it result from cleavage within the A4 segment catalyzed by secretase? Can give insight.   (i) Preparation of plasmid. Construction of plasmid for C-100 expression using two methods did. Each plasmid was individually identified as either PMTI 73 or PMTL 100. Will be.   Fabrication of PMTI 73: Digest commercial plasmid PUC-19 with EcoRI to remove polylinker Removed. Then, commercially available PWE16 was inserted into the digested PUC-19 to obtain PMTI 2300. Was. PMTI 2301 is BamHI / HindIII digested using oligonucleotide adapter Later derived from PMTI 2300. A to HindIII site of PMTI 2301 by blunt end ligation PMTI 2307 was prepared by inserting the EcoRI promoter fragment of PP.   The BamHI fragment from PC-4 (Kang et al., Supra) was used as a BamHI fragment of PMTI 2307. PMTI 2311 was prepared by ligating into the site. PM XhoI fragment from PC-4 PMTI 2312 was made by inserting it into the XhoI site of TI 2311. PMTI 2312 Cl 2.2 kb from EcoRI genomic clone of mouse metallothionein I gene at aI site  PMTI 2323 was created by inserting the BglII / EcoRI fragment. Mini gene PM Deleted the sequence between the KpnI and BglII sites of PMTI 2323 to create TI 2337 And using a synthetic oligonucleotide adapter sp-spacer-A4 The clones were ligated.   PMTI 2337 was cleaved with BamHI / SpeI and the resulting fragment was labeled with Bluescript KS (+) (Str PMTI 2371 by ligation to the BamHI / SpeI restriction sites of atagene). PMTI 2 Cleavage of 371 with HindIII / NotI 0.7 encoding the terminal 100 amino acids of APP695 The kb fragment was released. The sequence of the signal peptide was also encoded in the fragment. this The insert was ligated into the HindIII / NotI sites of pcDNAINEO (Invitrogen Corp.) The plasmid PMTI 73 was prepared.   Fabrication of PMTI 100: Includes full-length βAPP (PMTI 74) under the control of T7 promoter Cut KS Bluescript (M13 +) with BglII, fill in, then cut with EcoRV And religated, complete C-100 of βAPP without signal peptide A PMTI 90 containing segment was made. Cut PMTI 90 with XbaI / HindIII The 0.6 kb fragment encoding the terminal 100 amino acids of APP695 was released again, The plasmid PMTI 100 was constructed by ligating to the XbaI / HindIII sites of cDNAINEO. In each case, the vectors, inserts and plasmids were purified by methods known to those skilled in the art. Made.   (ii) Transfection and expression of C-100 fragment. Small scale of C-100 reference material Preparation for expression should be done 5 x 10 24 hours before use.^FiveHela S1 cells in 6 wells Each of the Coster plates (diameter 3.5 cm) It was started by inoculating the wells.   Mix equal volumes of crude virus stock solution with 0.25 mg / ml trypsin, then vortex vigorously. By kinetic stirring, sufficient vaccinia virus vTF7-3 was trypsinized, The cells were infected at a multiplicity of infection of 20 plaque forming units. Trypsinized The virus was incubated at 37 ° C for 30 minutes with vortexing every 10 minutes. Aggregation If clumps persist, incubate the incubation mixture at 0 ° C for 30 minutes in an ultrasonic bath. While sonicating. Repeat cooling sonication until no aggregates are detected again. I returned.   The trypsinized virus was then added to each well containing Hela S1 cells in 0.5 ml. Diluted with sufficient volume of serum-free DMEM to carry the virus. Aspirate the medium, Cells are then exposed to virus for 10 minutes with shaking to spread the virus. Infected for 30 minutes.   Approximately 5 minutes before the infection subsides, prepare a fresh transfection mix as follows: Do: 1 ml of OPTIMUM (Bethesd in a polystyrene tube with gentle mixing) a Research Labs, Gaithersburg, MD) 0.015 ml of lipofection reagent ( Bethesda Research Labs, Gaithersburg, MD) was added. Avoid vortex stirring. Then 3 μg of CsCl purified DNA was added and mixed gently.   The virus mixture was aspirated from the cells and then the transfection solution was introduced. Was. The resulting mixture was incubated at 37 ° C for 3 hours. Then on top of each well Overlay 1 ml of OPTIMUM, CO₂Incubate overnight at 37 ° C in an incubator did.   Cells were harvested by centrifugation 20 hours after transfection and 1% Trit on X-100, 10 μg / ml BPTI, 10 μg / ml leupeptin, 200 mM NaCl, 10 mM HEPES , 1 mM CaCl₂, Addition of 0.2 ml of lysis buffer containing 1 mM EDTA adjusted to pH 7.5 Lysate on ice Prepared. Complete lysis was monitored by light microscopy and collected immediately. Dissolution The solution takes less than a minute to complete, and the delay in this step causes the gelatinous mass to melt cause.   Recombinant lysates were stored at -20 ° C until further use. Preferably, the recombinant lysate is , Lacking 2-mercaptoethanol before freezing (3x) in SDS-PAGE sample buffer 1: Will be diluted 50.   Support for proteins produced by expression using either PMTI 73 or PMTI 100. Were compared by SDS-PAGE / immunoblot using APP-C terminal antibody (Fig. 2e). This study shows that PMTI 100 directs the expression of a single immunoreactive band, On the other hand, PMTI 73 directs the expression of two major bands of similar molecular size. Indicated. When applied to gels in higher amounts, PMTI 73 also produces thin bands of medium size. It was clearly visible (Figs. 2a-2d).   When a larger amount of PMTI 100 protein was applied to SDS-PAGE, a series of thin bands appeared. Appears (eg FIG. 2d). Other than the strong band of C-100 monomer with apparent Mr = 11.7kDa In addition, thin bands are observed at Mr = 25.5 kDa, 35 kDa and 45 kDa. this Et al. Due to the formation of dimeric, trimeric and tetrameric aggregates of the C-100 monomer, respectively. I do. An additional thin band of Mr 18.9kDa is also observed. Similar solutions with similar phenomena The report has been published in the literature (Dyrs et al., 1988, EMBO J., 7: 949).   The largest of the three bands produced by PMTI 73 is the PMTI 100. Was slightly larger than the single band observed. Maximum from PMTI 73 expression Amino acid sequence analysis of the band shows that the signal peptide sequence is cleaved from the first translation product. Has been shown to generate a C-100 fragment containing 5 extra amino acids at the N-terminus. .Embodiment 6 FIG. Manufacturing of immunochemical reagents   Three different immunochemical reagents were used in the present study:   (i) A rabbit polyclonal antiserum recognizing the C-terminus of APP was obtained and Produced by proteolytic processing according to the assay conditions described in Example 8. Used for immunoblot detection of the C-terminal APP fragment;   (ii) prepare an affinity purified antibody that recognizes the C-terminus of APP and Immunity for affinity purification of APP expressed in the culovirus-directed system Used to synthesize an affinity column (see Example 7); and   (iii) Mouse monoclonal antibody that recognizes the N-terminus of β-amyloid peptide And the C-terminus generated by proteolytic digestion of holoAPP695 Immunoassay for determining whether an APP fragment contains the full length β-amyloid peptide Used in the noblot assay (see Examples 9 and 10 for specific applications).   A method for producing each of the above three immunochemical reagents is given below.   (i) Rabbit polyclonal antiserum against the C-terminus of APP. Of human APP 695 Antisera were raised against the C-terminus, and Buxbaum et al., 1990, Proc. Natl. Aca d. Sci., 87: 6003. In the COOH terminal region of APP695 The corresponding synthetic peptide (hereinafter “βAPP645-694”) is available from Yale University, Prot. Obtained from ein and Nucleic Acid Chemistry Facility, New Haven, CT.   Immunize rabbits with βAPP645-694 to raise polyclonal antibodies I tightened it up. Large intestine expressing a fusion protein containing amino acids 19-695 of human APP695 Sera were screened by immunoblot analysis of fungal lysates. The recombination Sera that are immunoreactive with the fusion protein are serially transferred in vitro (Stra tagene ， La Kit purchased from Jolla, CA) and translation (purchased from Promega Corp., Madison, WI) Reticulocyte lysate kit) prepared from βAPP645-694 cDNA [³⁵S ] Further screened for immunoprecipitation activity against methionine labeled APP695 Was.   (ii) Polyclonal antibody affinity column for purification of holo APP.   Purification of synthetic C-terminal APP peptide immunogen: A having a cysteine residue at the N-terminus 80-90 mg of the crude synthetic peptide (P-142) containing the C-terminus of PP (649-695) was analyzed by HPLC. It was purified (yield 42%; 34 mg). Amino acid analysis, N-terminal sequence analysis and impact mass The laser desorption time for analysis was such that the purified peptide was cleaved at the N-terminal with the full-length peptide. It was shown to be a mixture of the isolated peptides (1/2 of the entire length was cut off).   Immunization of rabbits with purified P-142 immunogen: HPLC purified peptide APP (649-695 ) Was used to immunize a rabbit. Two rabbits in complete Freund's adjuvant An initial challenge with 125 μg of peptide was given, followed by an incomplete challenge at 3-week intervals. A booster of equal amount of peptide in Reutn's adjuvant was given. 14 over 9 months Blood was collected twice and optimal production of Ab was observed in blood at 16-32 weeks (vaccinia C100 and Western analysis using CHO APP). Antiserum blood in this interval 9 Pooled to an approximate volume of 0-100 ml.   Preparation of immobilized APP 649-695 affinity matrix for purification of antisera: 9.7mg of spirit Peptide APP (649-695) manufactured by BSA and Pierce Imject Activated Immunogen  It was conjugated to maleimide activated BSA using the Conjugation kit. Ellman reagent About 40% (3.88 mg) of the peptide bound to BSA as determined by. BSA binding Gel filtration of peptides (Purification buffer from kit = 83 mM NaH₂PO_Four pH 7.2, 900 Separated from unbound peptide by mM NaCl). Pooled from gel filtration column CNBr activity of 1g (3.5ml) of void volume (P-142 2.9mg / 12.5ml conjugated to BSA) Bound to derivatized sepharose (> 90% of the pellet by standard Pharmacia protocol). Bonded peptide conjugate). The remaining site was blocked with ethanolamine . The Sepharose affinity matrix was packed in a 1.0 × 3.5 cm glass column.   Rabbit polyclonal antibody using APP (649-695) affinity column Purification : Pooled mixed rabbit antisera from blood with optimal Ab production (100 ml / 3 .9g protein), wash buffer (100mM NaHCO_Three pH8.3, 750 mM NaCl) 1: 1 (v / v) dilution and peptide affinity color at a flow rate of 1.0 ml / min at 4 ° C. Loaded on the m. After loading (200 ml), wash the column until the A280 returns to zero. It was washed with (75 ml). The IgG was then eluted with 100 mM glycine pH 2.5 (40 ml). One fraction was collected in a tube containing 100 μl of 1.0 M Tris HCl pH 8.0. Neutralized The low pH IgG eluates were pooled (34 ml; 14.7 mg) and 1.0 l of 100 mM NaH at 4 ° C. CO_Three It was dialyzed against pH 8.3 and 500 mM NaCl.   Preparation of immunoaffinity column: cup of purified rabbit IgG on sepharose ring : 5.0 g of CNBr sepharose in 50 ml of coupling buffer (100 mM NaHCO 3_Three p H8.3, 500 mM NaCl) and dialyzed on Orbitron at 4 ° C for 21 hours Mixed with IgG. After coupling, the resin is placed on a glass filter to remove the coupling buffer. Rinse once with 100 ml blocking buffer (100 mM NaHCO 3_Three pH 8.3, 500 mM  Rinse 3 times with NaCl, 1.0M ethanolamine). Coupling buffer (100 ml ) Followed by two low pH buffers (100 mM NaOAc pH 4.0, 500 mM NaCl) (100 ml). For successive intermediate rinse steps and final rinse with coupling buffer (100 ml) Than, The resin preparation was completed. Coupling was 87% for a total of 17.5 ml of resin ( 0.727 mg IgG / ml resin).   (iii) Production and epithelialization of monoclonal antibody against β-amyloid peptide Tope mapping.   Hybridoma methodology. By multiple injections of a mixture of the following two synthetic peptides Balb / c mice were immunized: 1) AP of holoAPP695 containing β-amyloid P amino acids 597-638 (numbering according to Kang et al., Supra), and 2) C-terminal region APP295 amino acids 645-695, including. Standard procedure (Herzenberg et al., 1978, D.M. Weir Ed., Handbook of Experimental Immunology, pp. 25.1-25.7, Blackwell Scient ific Publications, Oxford, UK) using X63 / A for splenocytes from immunized animals. It was fused with g8.653 mouse myeloma cells. The supernatant from the resulting hybrid , Β-amyloid peptide immunogen bound to microtiter plates A was used to test for the presence of anti-peptide specific antibody. Used as an immunogen Cultures secreting antibodies that react with synthetic peptides were cloned twice by limiting dilution. And as described (Wunderlich et al., 1992, J. of Immunol. Met. hods, 147: 1) determined their isotypes. Cloned hybridoma cells Protein A Affinity Chromatography of Consumed Culture Solution from Serum-Free Culture Broth The secreted IgG was purified by Ruffy.   Epitope mapping: C2, one of the anti-peptide monoclonal antibodies The IgG2b, designated 86.8A, was synthesized by both EIA and immunoblot assays. It showed good reactivity with the adult β-amyloid peptide. Of this monoclonal antibody Epitope reactivity was determined by competitive EIA. Amino acid 597-6 of human APP695 12, 597-624, 597-638, 608-624, 621-631 and 645-695 (Kang et al., Supra. N-dansyl-Ile-Ser-Glu-Va as well as synthetic peptides containing l-Lys-Met-Asp-Ala-Glu-Phe-Arg-His-Asp-Asp-Asp-Asp (SEQ ID NO: 1), AP It was tested for its ability to inhibit the binding of C286.8A to P597-638.   The peptide recognized by the antibody is preincubated with the antibody in solution Then, if the β-amyloid peptide bound to the microtiter plate It will deplete the solution concentration of the antibody available for reaction. The result of such an experiment Is shown in FIG. 4 and described below.   APP597-612, 597-624, 597-638 and N-dansyl-Ile-Ser-Glu-Val-Lys -Met-Asp-Ala-Glu-Phe-Arg-His-Asp-Asp-Asp-Asp (SEQ ID NO: 1) only dose dependent It was able to inhibit C286.8A binding in a live manner. Those peptides are Amino acids 1-16, 1-28, 1-42 and 1-7 of the Lloyd sequence (residues of aspartic acid at the N-terminus) Numbering from the base). Β-Amyloid like APP645-695 Peptide without sequence sequence, or β-amyloid peptide sequence 12-28 or 2 Peptides containing 5-35 (APP608-624 and APP621-631, respectively) It did not inhibit the binding of the monoclonal antibody to the stock. These results are The reactive epitope for the clonal antibody is at least partly of human APP. It is shown to be present in the first 7 amino acids of the A4 region, namely APP597-601.Example 7. Purification of recombinant holo APP695   The first experiment of C-terminal processing of APP was CHO as described in Example 4. Performed with recombinant APP695 expressed in cells and by method 1 below Purified as described. With this substrate The characterization experiments described above are described in Example 8 below and C-terminal processing can be performed. Has led to the identification of six potentially different APP degrading enzymes.   Subsequently, it provides higher APP levels than can be achieved using the CHO expression system A baculovirus expression system was developed (see Example 4). From the baculovirus system Purification of Holo APP 695 of E. coli is described in Method 2 below. Purified baculois Ruth-derived holoAPP695 has the protease characteristics described in Examples 9 and 10 below. It was used to carry out an experiment.   All steps were performed at 0-4 ° C unless otherwise noted. Holo APP 695 is essentially Is an immunoassay using an anti-human APP695 C-terminal antibody as described in Example 8 below. Detected by blot analysis. Method 1. Purification of holoAPP695 from a stably transfected CHO cell line Made.   (a) Isolation of plasma membrane. From continuous culture of CHO cells in a roller bottle (see Example 4) Whole cell pellet (179 g) was collected by centrifugation (1500 g x 5 min) and salted. Sodium chloride (30 mM), magnesium chloride (1 mM), EDTA (10 mM), PMSF (200 μm / ml), E-64 (42 μg / ml) and pepstatin (3.8 μg / ml) in 50 mM Resuspended in Tris-HCl buffer pH 8.0 to a total volume of 600 ml. Cells with teflon sticks Homogenize (10 round trips) and then contain 41% sucrose and a protease inhibitor On 10 ml of homogenizing buffer lacking EDTA, PMSF, E-64 and pepstatin (25 ml per centrifuge tube). Centrifuge (26,800 RPM x 60 minutes, Beckman SW-28 After removing the interfacial layer, carefully remove the interface layer (total volume approx. Dilute with genize buffer (lacking protease inhibitor), Teflon stick (3 round trips) Re-suspended in, and recentrifuged as above, tightly plugged Pellets were obtained. Decant the supernatant and pellet with 50 mM Tris-HCl pH 8.0. The Ret was resuspended in a total volume of 100 ml (3 rounds with a Teflon rod). Centrifugation (50,000 RPM x 60 minutes, in a Beckman 70 Ti rotor) to obtain a pellet, which is 50 mM Tris-HC l, resuspended in a total volume of 57 ml in pH 8.0.   (b) Solubilization of plasma membrane. 37 ml of the above resuspended CHO plasma membrane preparation was 20% (v / v) Triton X-100 stock solution with a mixture of protease inhibitors to give concentrations EDTA (1 mM) in the above homogenization buffer (45 ml total dissolved volume). , E-64 (24μg / ml), PMSF (53μg / ml), Pepstatin A (11μg / ml) and Tr iton X-100 (2.2% v / v, final concentration). The mixture was shaken gently at 4 ° C. for 30 minutes Afterwards, the insoluble material was centrifuged (50,000 RPM x 40 minutes in a Beckman 70 Ti rotor). Removed. Pass the supernatant containing solubilized holo APP through a 0.45 μM disc filter. And filtered.   (c) Purification of solubilized holoAPP695 by strong anion exchange chromatography. The above supernatant containing holo APP695 was diluted with an equal volume of distilled water, and 0.1% Triton X-100 was added. Mono-Q HR 10/10 column pre-equilibrated with 20 mM Tris-HCl buffer pH 8.0 containing Applied to Once loaded, add a direct volume of 0-1 M NaCl in a total volume of 210 ml equilibration buffer. The column was eluted using a linear gradient. The flow rate was maintained at 3 ml / min throughout. 17 ~ 22 It contains most of immunoreactive APP695 which elutes in the electric conductivity region of mmho (4 ° C). Proteins together, then 2 liters of 5 mM Tris-HCl containing 0.025% Triton X-100  Dialyze against pH 8.0 for 4 hours and centrifuge (26,800 RPM x 60 minutes, Beckman SW 28 A slight turbidity was clarified by (in the rotor).   (d) Heparin-agarose chromatography. Cleared sample is dialyzed into buffer Heparin-agarose color pre-equilibrated with liquid Applied to the frame (15 x 1.6 cm). When loaded, a light brown band in the upper third of the column occured. Once loaded, 5 fractions were collected (1 ml / min flow rate was used throughout). Next Adjust sodium chloride to the following final concentrations: 0, 150, 300, 600 and 2000 mM The column was eluted stepwise with 85 ml of the equilibration buffer prepared above. Immunodetectable holo Most of the APP elutes at 600 mM NaCl, the next fraction is at 300 mM Recorded in. APP collected at 300 mM and 600 mM NaCl was collected separately, Aliquots were stored frozen at -80 ° C. The APP used in the next experiment is a 300 mM image. It was from the minute. Partially pure from 300 mM heparin agarose eluate The APP yield was 5.5 μg per gram of wet CHO cell pellet (Bradford Assay). It was). The APP in this preparation is approximately 25% pure based on SDS-PAGE analysis. It was rated as Degree. Method 2. Holo APP695 and holo from recombinant baculovirus-infected insect cells Purification of APP695ΔNL.   (a) Solubilization of cell pellet. Combine the cell pellets from the two 5 liter fermentations (Total detectable protein 8.9g), the following inhibitors: Pepstatin A (25μg / ml ), Leupeptin (25 μg / ml), chymositatin (25 μg / ml), antipain (25 μg / ml), aprotinin (25 μg / ml), benzamidine (4 mg / ml), PMSF (0. 87 mg / ml) and 160 ml of 0.32 M sucrose containing EDTA (25 mM), and Homogenized with a Teflon rod (10 round trips). Centrifuge the homogenate (105,000g X 1 hour in a Beckman 70 Ti rotor), then 0.5M NaCl and the same concentration as above. Teflon rod (10 round trips) in 160 ml of 10 mM Tris-HCl buffer pH 7.5 )) And resuspended the pellet. Short sonication (Branson Sonifier Cell , After 2 minutes at power level 4), add Triton X-100 to a final concentration of 5% (v / v) and suspend. The solution was gently stirred at 4 ° C for 20 minutes. The mixture is then centrifuged (50,000 RPM x 60 minutes , Beckman 70 Ti rotor), the first supernatant (574 mg protein) with heparin- Carefully removed for agarose chromatography. Pellet teflon stick (20 round trips) with 10 mM Tris-HCl buffer containing 0.5 M NaCl and the above concentrations of each inhibitor The suspension was resuspended in 160 ml of liquid pH 7.5. 5% (v / v) Triton X-100 as described above Solubilization with a second solubilization supernatant (683 mg protein). Quality).   (b) Radiant flow chromatography on heparin-agarose. Obtained above The two supernatants were separately purified on heparin-agarose as follows. 3.5l capacity Volume of purified water and 1 M Tris pH 9.5 to a conductivity of 1.8 mmho and pH of 8.0 Dilute the supernatant by addition and fill with 250 ml of resin and 0.1% Tri Superflow 25 pre-equilibrated with 5 mM Tris-HCl buffer pH 8.0 containing ton X-100 It was applied to 0 column (Sepragen). Once loaded, wash the column with 3 liters of equilibration buffer. Purified and then eluted with equilibration buffer containing 600 mM NaCl. Flow rate of 30 ml / min from beginning to end used. Fraction (45 ml) at A280 nm, total protein (Bradford assay), and Detected by immunoblot against anti-APP-C terminal antiserum of Example 6 (i) Were monitored for levels of immunoreactive APP. Image containing significant APP The minutes were combined and subjected to antibody affinity chromatography.   (c) Antibody affinity chromatography. First supernatant (276 mg protein Quality) and second supernatant (containing 113 mg protein) heparin-agarose The 600 mM elution pools from the above purification were combined, adjusted to pH 8.3, and used in Example 6 (i Sepha as described in i) Comprises an affinity purified C-terminal antibody coupled to sucrose, and 500 mM NaCl, 100 mM sodium bicarbonate buffer containing 0.1% Triton X-100 pH 8.3 Apply to an antibody affinity column (10.5 x 1.5 cm) that has been pre-equilibrated with Was. Chromatography was always performed at a flow rate of 1 ml / min. Once loaded, load the column Wash with 70 ml equilibration buffer, then 100 mM glycine buffer with 0.1% Triton X-100 Elution was carried out with 50 ml of liquid (pH 2.4). Fraction (5 ml) was added with 0.5 ml of 1M Tris-HCl pH 8.0 at 280 nm, total protein (Bradford assay) as above. ), And the presence of immunodetectable APP. Significant AP Fractions containing P were combined. Combine using the combined heparin-agarose eluate. The affinity purification procedure was repeated 5 times in total. Each recovered from successive purifications The combined APP pools yielded a total of 9 mg of APP.   (d) Strong anion exchange chromatography. Antibody affinity chromatograph The combined fractions (9.0 mg protein) from E.T.E., 0.025% (v / v) Triton X-10 Mono-Q pre-equilibrated with 20 mM Tris-HCl buffer pH 8.0 containing 0 and 150 mM NaCl Applied to HR 5/5 column. Once loaded, a linear gradient of 0.15 to 1M NaCl in a total volume of 70 ml Was used to elute from the column. A flow rate of 0.5 ml / min was used throughout. Significant immunoassay Fractions containing ready-to-use APP were combined and aliquoted until use It was stored frozen at -80 ° C.   ΒAPP migrating as a single band (Mr 110 kDa) on SDS-PAGE (starting twice) 5.6 mg from a 5 l fermentation experiment), the amino acid composition showing 86% agreement with the theoretical composition, Expected N-terminal of mature protein (Leu-Glu-Val-Pro-Thr-Asp-Gly-Asn-Gly-Le u-). Purification of the βAPP695ΔNL protein was essentially as described above. there were. The final preparation (0.3 mg from two starting fermentation experiments) had a theoretical value of 70% The only contaminant with a consistent composition and not βAPP related on SDS-PAGE (Mr 64KDa) was included. Both forms of purified βAPP on immunoblot Was reacted with a rabbit polyclonal antibody against the C-terminal region of βAPP.   Amino acid analysis was performed essentially as described elsewhere (Dupont, D.R. , Keim, P.S., Chui, A.H., Bello, R., Bozzini, M. and Wilson, K.J., "A. comprehensive approach to amino acidanalysis ”, Techniques in Protein Ch emistry, Tony E. Hugli, Academic Press, 284-294 (1989)). 2.0% Feno Hydrolyze the sample for 2 hours at 160 ° C under argon in the gas phase with 6N hydrochloric acid containing I understand. Phenylthiocarbamoyl amino acid analysis is based on the online type 130A separation system. Applied Biosyste with Stem and Nelson Analysis Model 2600 Chromatography Software It was performed on the ms 420A Derivitizer.Embodiment 8 FIG. Detection of APP695 degradation catalyzed by human brain protease subfractions Immunoblot assay for   (a) Incubation with substrate APP   (i) A 5 μl aliquot of the ion exchange fraction (obtained from the steps described in Example 1) ) Or a concentrated pool of fractions (Example 3) for addition of the required amount of 2M stock buffer. Recombinant human APP695 (10.75) adjusted to a final concentration of 140 mM MES buffer pH 6.5. μl) and incubated at 37 ° C. for 24 hours. Final in incubation solution The buffer concentration was 95 mM, pH 6.5. Of APP695 during the incubation time Proteolysis occurs, producing smaller Mr fragments.   (ii) Proteolysis reaction was performed using the following 3 x Laemmli SDS-PAGE sample Terminated by addition of 7.5 μl of buffer: 36% (v / v) glycerol, 12% (v / v) SDS, Trace 10% (v / v) 2-mercaptoethanol and trace amount of bromophenol blue 1.5M Tris-HCl, pH 8.45 containing dye for use. Heat the sample (87 min at 100 ° C), then And cooled.   (b) SDS-PAGE analysis   The reaction mixture (15 μl) was applied to a 10-20% acrylamide gradient Tricine gel (normal thickness 1 .0 mm, 15-well Novex Precast Gel, Novex Experi-mental Technology, San Die go, CA). Gel at 50 V until the sample enters the gel under constant voltage conditions Was run, and the voltage was raised to 100V once in the gel. Trace dye at bottom of gel Electrophoresis was stopped when it reached within 0.5 cm. Pre-stained in the range of 3-195 kDa Used Mr marker (Bethesda Research Laboratories, Gaithers-burg, MD) A calibration curve for the gel was prepared. 10 μl each of kit containing high and low molecular weight markers Mix with 10 μl of 3 × sample buffer and treat as described in section (a) (i). The following molecular weight marker proteins are included in the kit as pre-stained markers : Myosin H chain (196 kDa); Phosphorylase B (106 kDa); Bovine serum albumi (71kDa); ovalbumin (45.3kDa): carbonic anhydrase (29.1 kDa); β-lactoglobulin (18.1 kDa); lysozyme (14.4 kDa); Psin inhibitor (5.8kDa); insulin A and B chains (3kDa).   (c) Immunoblotting   (i) The gel was then loaded onto a mini-trans blot electrophoresis cell (Biorad Labs, Richmon d, CA). The following migration buffer maintained at 4 ° C: 150 mM glycine and 20% (v / v ) ProBlott with 20 mM Tris-HCl buffer pH 8.5 containing methanol^TMMembrane (Applied  Electrolyze protein on Biosystems, Foster City, CA for 1 hour at 100V (constant pressure) Blotte I did it.   (ii) Remove the ProBlott membrane and add it to 15 ml of blocking buffer of the following composition at room temperature: Leave for 1 hour: 5% (w / v) in 10 mM Tris-HCl buffer pH 8.0 containing 150 mM NaCl Skim milk powder.   (d) Immunodetection of APP and C-terminal degradation products   Rabbit Polyclones Raised Against Synthetic Human APP695 C-Terminal Peptide Immunogen Transfer the membrane to 15 ml blocking buffer containing 1: 1000 dilution of the internal antiserum, And it incubated at 4 degreeC overnight.   Membrane the membrane 3 times for 5 minutes each with 15 ml of blocking buffer with gentle shaking. Rinsed. Then goat anti-rabbit IgG (Fisher Scien) conjugated with alkaline phosphatase membranes in 15 ml blocking buffer containing 1: 1000 dilution of Tific, Pittsburgh, PA). Transferred and incubated at room temperature for 90 minutes. Then 15 ml each time with gentle shaking Membranes were rinsed three times in succession for 10 minutes with the blocking buffer of.   Then each is 100 mM NaCl and 5 mM MgCl₂Containing 100 mM Tris-HCl pH 9.5 containing Consisting of 15 ml of alkaline phosphatase buffer solution each time, the membrane is continued 3 times for 5 minutes each. Washed. The gel is then mixed with 15 ml of 100 mM NaCl, 5 mM MgCl 2.₂Containing 100 mM Tris HCl p H9.5, and 50 μl BCIP substrate (50 mg / ml, Promega, Madison, WI) and 99 μ The gel was incubated with 1 NBT substrate (50 mg / ml, Promega) in the dark. Until no further enhancement of low molecular weight immunoreactive bands is seen (typically chamber Incubation was continued for 3 hours at warm temperature. Then rinse the gel with deionized water, Dried. Fractionated human AD skin enzymatically cleaving APP695 to produce a C-terminal fragment Analysis of Quality Mono-Q Pool Ability   Each of the P2, S and M pools described in Example 3 was loaded with the immunoblot assay described above. I went to sleep. Use and combination of authentic APP substrate molecules The specificity of the immunological detection method applied to APP in relatively crude biological extracts It provides a selective method for detecting the activity of degrading enzymes, and provides highly purified enzyme preparations. Avoid the need to use. Therefore, some partially purified pools are time-dependent. It has a proteolytic activity capable of forming C-terminal APP fragments in a live manner Was. For P2V (panel a), MIII (panel b) and SI (panel c) And human A for pre-pooled individual fractions of the P2VII pool (panel d) A typical example of an immunoblot analysis of D brain is shown in FIG.   For example, a time course experiment as shown in FIG. The fragment was shown to be absent in the substrate or enzyme fractions at time zero. In addition, the substrate Incubation alone did not result in the formation of those fragments (see, eg, Figure 2a). Lane 2, lane 2 of Figure 2d, lane 8 of Figure 2f). The size area of the above band is Depending on the enzyme fraction, Mr = about 11.5 kDa to 25 kDa, but other formed during the reaction The number of products was surprisingly low. At pH6.5, out of a total of 39 AD pools Eight were found to have such activity. Those pools are i) brain miniatures Min, ii) ionic strength of column elution, and iii) qualitative APP cleavage pattern. It was possible to distinguish from each other.   Six selected pools (“M-III, M-VIII, S-I, S-III, P2-V And P2-VII ") contained significant APP degrading activity. versus The corresponding control brain pool also contained some of the above activity, but the level of activity was It was not possible to determine if they differ between the AD pools. The above 6 Each of the enzymes has an enzymatic activity capable of forming a 11.5 kDa C-terminal APP fragment. I had.   The proteolytic product of Mr 11.5 kDa was found to be the major Is a C-terminal product detectable by epidemics and Starting from the N-terminal aspartic acid of the Lloyd peptide to the C-terminal of the full length molecule With a recombinant C-terminal fragment of APP (C-100 fragment) which will contain the open reading frame We paid particular attention because it was found to move. This simultaneous movement is illustrated in Figure 2d. You. This means that the 11.5 kDa band is at or near the N-terminus of the A4 region. Is a product of endoproteolysis of, and fragments of The above-mentioned protease activity capable of forming a Implies that it may play a role in vivo.   FIG. 2d shows 11.5 kDa of APP proteolysis, at least for P2 pool VII. It shows that the C-terminal degradation products can aggregate. PMTI 100 Directive with C-100 reference material In addition to the migrating Mr 11.5 kDa peptide band and the appearance of the 18 kDa fragment, Mr 24.3, Another fragment appears at 27.4 and 35.5 KDa. This 24.3kDa and 35.5kDa band Has the Mrs expected for the dimer and trimer of the C-100 fragment, respectively, and It moves almost together with the corresponding thin band in the C-100 fragment, which is due to the collection ( See Example 5 for details).   2a-2c also show the assay for examining the effects of classical protease inhibitors. Help prove that is available. For example, from Figure 2a, P2-V is meta Inhibited partially by Nol and completely by methanolic pepstatin A While M-III (Fig. 2b) and S-I (Fig. 2c) both Is also completely inhibited by aprotinin and cystatin. Therefore, the assay is In one aspect, it applies to the search for new in vitro inhibitors of APP degrading enzymes. in addition The potential compound thus identified is a suitable animal model such as APP or its Designed to overexpress a β-amyloid-containing fragment of In vivo using animal animals Tested for efficacy.   Table 4 below shows the six major pools of APP degrading activity recovered from the Mono-Q fraction. Properties of peptide (peptide product size, apparent pH dependence on product formation) , And the effects of commercially available protease inhibitors, etc.).   Some activities have an optimum pH in the alkaline region and the action of lysosomal cathepsins It didn't seem to depend on you. Several researchers have found that pathological APP processing is It is reported to be carried out by a protease in the dosome-lysosomal pathway (Ca taldo et al., 1990, Proc. Natl. Acad. Sci. USA, 87: 3861; Benowitz et al., 1989, Ex perim-ental Neurology, 106: 237; Cole et al., 1989, Neurochem. Res., 14: 933) Therefore, this observation is important. Enzymes in this pathway show acidic pH optimum Would be expected.   Based on the available data, M-III and S-I meet all of the listed criteria. And very similar, probably cross-contaminated in each of the S and M fractions. Would represent an enzyme. Therefore, probably the human brain decomposes APP into 11.5 kDa C- A minimum of 5 different protease activities capable of producing 100-like product fragments Will include.   Table 4 shows that some of the activities are insensitive to inhibition by any of the tested inhibitors. There are, and those enzymes may represent members of an unusual group. M The activities involved in the formation of the 11.5 kDa C-100 fragment in -III and SI are Is either of the cysteine type or represents a member of a rare group. Alternatively, those fractions contain both serine and cysteine proteases. , Where both enzymes play an essential (sequential) role in the production of C-100. Add P2-V has aspartic protease activity and serine protease activity. Contains both. P2-VII is based on the sensitivity to PMSF ZE activity is contained. However, in subsequent studies pepstatin inhibitory activity Was also observed, and aspartic acid pro- tein was detected in P2 along with the serine protease activity shown in Table 4. Indicates that thease coexists. Enzyme activity in S-III or M-VIII was tested It was not sensitive to any of the inhibitors. In which case The enzyme pool was also incubated with the N-dansyl peptide substrate used in Example 3. It was not possible to prove inhibition of APP degradation by beating.   Recovery of APP activity (Example 8) and peptidase activity of Mono-Q pool (Example 3) And comparison with Figure 1) clearly shows that there is little correlation between both activities. You. For example, in a pool showing relatively low peptidase activity for APP degradation activity Was contained in large amounts. It is a peptidase with poor APP degrading activity, Requires a complete folded APP substrate for activity, or also (likely Selected peptide is not suitable for pathological APP processing Suggest that it may represent a position. Anyway, this finding is why other researchers Identification of common APP-degrading enzymes using a peptidase substrate-based assay Explain if and were unsuccessful.   From the above considerations, the assay of the present invention was used to isolate a specific APP degrading enzyme from human brain. It is concluded that it has sufficient specificity to allow   In a further study, we will follow the recovery of P2-VII related APPase. An immunoblot assay was used. This pool is characterized by 6 active The most abundant of these, and the C-terminal fragment that seems to be amyloid is produced. Studies were concentrated in this pool as they were made (Fig. 2d). It ’s the P2 subdivision Io. Represents the main activity that can be recovered from the exchange separation and is consistent with the main peak of peptidase activity. It was eluted at some point in the gradient that was not.   P2-VII fractions representing APPase were pooled and two tandem Superose Subjected to size exclusion chromatography on 12 columns (Pharmacia). Elution image Minute Peptidases and APPases The activity was analyzed (Fig. 3a). Although the KM cleavage activity seems to overlap partially, , MD activity peak again did not coincide with the APPase peak. Known molecule The chromatographic calibration for the quantity marker For ze activity, an apparent median Mr value of 31.6 kDa with a variation of ± 6.5 kDa was given.Embodiment 9 FIG. Identification of cathepsin D as an APP-C terminal processing enzyme   Of the baculovirus expression system described in method 2 of example 4 and method 2 of example 7 A larger amount of holoAPP695 was obtained by using the purification scheme, so The content of APP degrading enzyme in the pool of fractions generated based on the peptidase activity Rather than assessing (as done in Example 8), Allows tracking of APP degrading enzyme recovery in individual column fractions from purification became.   For each Mono-Q fraction from the purification of the solubilized P2 fraction according to the method of Example 1 The contents of all APP degrading enzymes are shown in FIG. Mono-Q chromatography Compared with similar analyzes of the soluble (S) and microsomal (M) fractions The relative staining intensity of the elementary C-terminal APP fragment was consistently highest in the P2 fraction from Mono-Q. Met. APP degrading activity in P2 is shown as two separate migration peaks from Mono-Q. Recovered (peaks A and B in Figure 5).   Peak A is a load and low ionic strength wash, ie the P seen in our earlier studies. Elution occurred in a region that closely matched the recovery of 2-V (Table 4), while peak B was Previously P2-VII activity was observed (Table 4) and serine protease activity and The pooled region and the heavy region shown to comprise both Duplicate. peak Degradation products of similar size with both A and B activity have Mr = about 28, 18, 14 and < Although observed at 11 kDa, the relative staining intensity of the 18 kDa band is higher than that of peak A. B was much bigger. Peak B was pooled and as described in method 1 of Example 1 It was subjected to purification on Superose 6HR. The elution fractions have overlapping elution curves. It contained two qualitatively different types of activity. Observed in the original Peak B fraction (Fig. The activity that produced an APP degradation pattern that most closely resembles that of 5) was found in the gel filtration fraction. Recovered at 51-56 (FIGS. 6b and c), which is consistent with an apparent Mr of 15-25 kDa. . This elution peak precedes the elution of activity that preferentially forms 18 kDa degradation products. It happens and is probably catalyzed by a larger apparent Mr protease. This The latter activity of S. cerevisiae was probably due to the presence of the serine in the P2-VII pool of Table 4 of Example 8 above. Equivalent to protease. Active fraction from gel filtration purification of peak B (15-25 kD active fraction of Mr region of a) for inhibition by classical protease inhibitors (FIG. 7). Those studies are measured by quantitative inhibition by pepstatin A. And peak B activity is mostly catalyzed by aspartic proteases Confirmed.   There are relatively few known human aspartic proteases. Have been identified Those include cathepsin D and E, renin and pepsin. I It is possible that the activities observed by each may correspond to some of those enzymes. To test its potency, the ability to enzymatically degrade baculo-derived holoAPP was examined. Commercially available human renin (Calbiochem, San Diego, CA Catalog No. 553864) and Human Cathepsin D (Human Liver, Calbiochem, San Diego, CA Catalog No. 219401 ) Examine the preparation.   Human cathepsin D (catalog number 219401, Calbiochem, San Diego, CA) Electrophoretic homogeneity on SDS-PAGE developed by silver staining (Apparent Mr = 29 kDa under reducing conditions), and has a good theoretical composition of cathepsin D. It had an amino acid composition that showed concordance (93%). All N-terminals match cathepsin D However, the major sequence present in equimolar amounts is that of the mature protease light chain (GPIPEVLKNY). Corresponds to the heavy chain (GGVKVERQVF).   Renin was inactive (not shown), but cathepsin D was up from Mono-Q. C-terminal degradation formation with a pattern similar to that observed for P2 peak B (FIG. 5) described above. APP was selectively cleaved to yield the product. Thus, commercially available cathepsin D The preparation degraded holoAPP in a time-dependent manner to yield major C-termini of approximately Mr 18 and 28 kDa. The product was given. Inhibition of activity by pepstatin A is due to cathepsin D in the reaction. Was confirmed (Fig. 8).   We obtained a commercially available polyclonal antibody against human cathepsin D (Dako Corp, C arpinteria, CA, Catalog No. A561), which is a human cathepsin on an immunoblot. It was found to be immunoreactive with Mr 27-28kDa, which is reactive with γ-D. I guessed. P2, from Mono-Q purification of either soluble or microsomal fractions To determine whether the chromatographic fraction contains immunoreactive cathepsin D This antibody was used in an immunoblot assay. This antibody is It did not cross-react with human renin.   A significant amount of cathepsin D was detected in the P2 fraction (FIG. 20d) having APP degrading activity. And the soluble fraction (data not shown) were observed in the Mono-Q fraction. Interesting thing And in the analysis of the P-Mono-Q fraction, each corresponds to peaks A and B (Data not shown) Two chromatographically different cathepsin D reactivities Peaks were occasionally observed. Immunoreactive aspartic acid p-related to peak A activity Rotase cathepsin D was previously identified by P2-V in Example 8. Matched the marked area. This is because peaks A and B are in multiple forms of cathepsin D. Suggest that it may be due to Multiple forms of cathepsin D are described elsewhere And is due to differences in post-translational modifications of single gene products. P2 peak B Immunoblot analysis of gel filtration column fractions from further purification (FIG. 5) showed APP The presence of a peak of cathepsin D immunoreactivity that closely matches the peak of degradative activity. Indicated.   Cathepsin D co-migrates with immunoreactivity and AP as well as cathepsin D Peak B protease further purified by gel filtration in addition to degrading P Has the same optimum pH (pH4−) as cathepsin D for the formation of C-terminal APP degradation products. 5) and ionic strength dependence (Fig. 9). Finally, observe in the P2 fraction The immunoreactive bands generated were analyzed on the BioRad Miniphor chromatography system. When subjected to preparative IEF of p. 4-6) (data not shown). Overall, those data are human Pepstatin-sensitive APP protease activity observed after Mono-Q fractionation of brain P2 Strongly supports the fact that is due to the action of cathepsin D.   Peak B protease (purified by gel filtration) and cathepsin D peptidase Synthetic peptide: N-dansyl-Ile-Ser-Glu-Val-Lys-Me t-Asp-Ala-Glu-Phe-Arg-NH₂(SEQ ID NO: 7) was used for comparison. Both enzymes are extremely The peptide was hydrolyzed in a time-dependent manner despite the very slow rate. Both enzymes A major cleavage was observed at the -Glu-Val- bond, and at the -Met-Asp- bond. A small degree of cleavage was observed at (Fig. 10). In Figure 10, the 24-hour time point depicted By the time most of the Met-Asp cleavage products were further converted to Glu-Val cleavage products. Note. As expected, cathepsin Both peptide reactions catalyzed by D and P2 enzyme preparations lead to pepstatin A. More inhibited.   Both enzymes show an acidic optimum at pH 4 for hydrolysis at the -Glu-Val- bond. (Fig. 11). -Met-Asp- bond hydrolyzed with cathepsin D at an acidic optimum value (<pH3.0) However, two optimum values (pH3.0 and pH7.0) were observed for the P2 enzyme, which was Involvement of additional contaminating P2 protease with possibly neutral pH optimum in the reaction This is probably because (Fig. 11). Cathepsin D normally hydrolyzes between hydrophobic residues. I However, at acidic pH values, the protonated (neutral) form of Asp and Glu side chains Is it sufficiently hydrophobic to satisfy the subsite binding conditions of the protease? It may happen. The pKa of the Asp side chain is more acidic than the Glu residue, and investigated in Example 11. It will protonate to a lesser extent than Glu residues throughout the pH range. This is -Glu-Va The rate of cleavage by cathepsin D at the -Met-Asp- bond is higher when compared to the l- bond. Explain smaller, and the optimal pH for cleavage at this site is lower (<pH Explain the hint 3).   Further use of monoclonal antibody C286.8A in an immunoblot assay Studies have confirmed that P2 enzyme activity is due to cathepsin D. More recently of us In the present study, this activity was demonstrated by ion exchange chromatography of the solubilized P2 fraction. Recovered as a single peak eluting over a fairly narrow fraction at the beginning of the salt gradient. (Fig. 20). This activity hydrolyzes APP 695 in vitro to give a 10-38 kDa cycle. Generated fragments (Fig. 20b), all of which were the first 7 of β-amyloid. Reacted with a mouse monoclonal antibody (C286.8A) that recognizes N-terminal residues ( (Fig. 4). The activity of the pooled fractions is aspartic protease inhibitor 10 Completely inhibited by μM pepstatin A, EDTA (1 mM), PMSF (0.4 mM), E-64 (0.1 mM) and other protease class inhibitors such as aprotinin (10 μg / ml) Less affected (not shown). APP degradation activity (Fig. 20b) N-Dansil-ISEVKMDAEFR-NH₂Hydrolyzes at the -E-V- bond Consistent with elution of pepstatin-sensitive protease (Fig. 20a). Cathepsin D , 20kDa cathepsin D light chain as assessed by holoAPP And elute at the same time as the peptide degrading activity.   High-purity human cathepsin D was pooled from Mono-Q chromatography to P2A. Distinguishable from that observed using spargate protease activity (Figure 21b). Holo A to generate a pattern of C286.8A immune reaction products (FIGS. 20b and 21b). PP was decomposed. Immunology between P2 aspartate protease and cathepsin D Immunosorbent experiments were performed to examine the possibility of physical identity. Their APP decomposition The ion-exchange fractions pooled on the basis of activity (Fig. 20) were collected against cathepsin D. Columns of standardized affinity purified antibodies or controls containing purified control IgG Equal volume was applied to either of the columns. The A280 elution curves of the two columns should not overlap. (Fig. 21a). The flow through from the control column was collected after the first interstitial volume (immediately). (FIG. 21b, fraction 5 onward) contained the same level of APP degrading activity as the applied pool. In contrast, APP-degrading activity was measured by anti-cathepsin D up to fraction 9 (5.7 void volume). It is essentially absent in the flow-through fraction from the rum and is applicable to fractions 27 (31 void volume). It did not reach the same level as the one in the game. Easy access from anti-cathepsin D column The absence of APP-degrading activity in the fractions indicates that the same anti-cathepsin D Immunoreactive cathepsin D light chain (20 kDa) and heavy chain (27 kDa) detected by the body ), Which coincided with the depletion (Fig. 21c). Cathepsin D immunoreactivity is 0.5% Triton X-10 Using 100 mM glycine pH 2.5 including 0 Recovered from the anti-cathepsin D column by elution, but recovered from the control column Did not. Tampas other than those corresponding to immunoreactive cathepsin D in this eluate No quality band was detected (Fig. 21c). Therefore, the absorbed APP Degradation activity was derived from immunologically cross-reacting proteases other than cathepsin D The possibility is almost unthinkable. Unfortunately, the pole from the anti-cathepsin D column Traces of APP degrading enzyme activity were recovered (data not shown). This is probably Probably because the activity was inhibited by the neutralization elution buffer. It quantitatively inhibited the degradation of holoAPP by cathepsin D (not shown).   Immobilized polyclonal antibody to human cathepsin D was also added to the ion exchange pool. Immunosorbing the APP peptide hydrolytic activity present in the cell (Fig. 22). Partial addition Under conditions of degradation, the ion exchange pool will damp at both the M-D and E-V bonds. Sill-ISEVKMDAEFR-NH₂Was cleaved (the long-term incubation Under conditions, the product from M-D cleavage ultimately produces E-V due to secondary proteolysis. Was converted to a thing). Similar to APP degrading activity, EV (Figure 22a) and MD (shown) Peptidase activity resulting in a fluorescent product of the bond cleavage from the control IgG column. It first appeared in Fraction 5 of the flow-through fraction of the It was essentially lacking up to ~ 13 (Figure 22a). Then use glycine / Triton pH 2.5. The resulting elution resulted in low levels of M-D and E-V peptidase activity with anti-cathepsin D color. But not the control IgG column (FIG. 22b). Straightforward Each of the peptidase activities recovered in the fraction It was completely inhibited by statin A. APP seen in so-called Swedish FAD Corresponding peptide that mimics the locus (N-dansyl-ISEVNLDAEFR-NH₂)use Tsu Similar results were observed in parallel experiments (FIGS. 22c and 22d). However However, in this latter peptide, the major fluorescent product that accumulates is from the LD bond cleavage. occured. The accumulation rate of this peptide is so fast that the substrate available for this reaction All was well consumed within the incubation time and the difference in the flow through fraction in Figure 22c. Causes underestimation of activity. It can also be collected in the flow-through fraction or by elution. Peptidase activity was inhibited by pepstatin A. This peptidase Faster apparent speed than observed for MD bond cleavage in wild-type APP mimetic substrates Note that in some degree the L-D bond present in the Swedish mimetic peptide was hydrolyzed .   Further experiments show that the peptide bonds in APP cleaved by cathepsin D are identical. The sex was investigated. More APP was subjected to cathepsin D hydrolysis at pH 5.0. Limited proteolysis under non-denaturing conditions was used. Incubation mixture Analyze by SDS-PAGE, immunoblot, then isolate individual product bands Anti-β-amyloid by Coomassie blue staining or as described in Example 6 (iii) It was localized by immunodetection using a monoclonal antibody. In coomassie blue The localized major band was subjected to N-terminal sequence analysis.   Figure 12 shows a Coomassie blue stained blot of such reaction mixture as well as an immuno Both blots (with anti-β-amyloid monoclonal antibody) are shown. Contrast In the absence of cathepsin D (in this case, after addition of SDS-PAGE sample buffer) Cathepsin D is added to the incubation mixture) or APP695 substrate Incubation was also performed in the absence. Complete incubation mixture Eight major product bands were observed by Coomassie blue staining (Fig. 12a) of , Those bands were missing in any of the controls above. All of those bands Some, but not some, are β-amyloid peptides. Anti-A4 monoclonal antibody that recognizes the epitope in the first 5 residues of peptide (Fig. 12c). N-terminal sequence analysis of Coomassie stained products is shown in the table below. Given the array to be raised.   As expected, what was reported about cathepsin D hydrolysis of other substrates (Moriyama 1980, J. et al. Biochem., 88: 619). Product was observed. With the exception of the reported cathepsin D specificity, band 3 -Glu-Val-cleavage forming a major product and a by-product of band 5, as well as bands 1 and It contained the -Leu-Arg-cleavage product of nd2 (Table 5).   Cleavage of the -Glu-Val- bond of APP593-594 was observed in pepstatin inhibitory reactions. The ability of cathepsin D to cleave the corresponding bond in the observed peptide substrate (see above) ). Cathepsin D normally hydrolyzes between certain hydrophobic residue pairs. Gl Cleavage of the u-Val bond is surprising, but probably under acidic (pH 5) conditions. Of the protonation of the side chain (pKa = 4.25) of the glutamic acid residue that neutralizes the mic acid residue It will happen because.   In fact, it can be calculated that at pH 5.0, 18% of -Glu- side chains will be protonated. it can. Such acidic conditions may occur in lysosomes, secretory granules, or It can be induced at the time of tissue injury or after hypoxia or local absence.   Most importantly, cathepsin D is the N-terminal of the normal form of β-amyloid. At the N-terminal side of the -Glu-Val-bond by 3 amino acid residues from the -Asp- residue Atypical hydrolysis produced a 5.6 kDa product (band 3, Table 5). You. This fragment is a blot taken from incubation without cathepsin D Was not present in the equivalent part of. Furthermore, this fragment is a full-length β-amyloid peptide It is of a suitable size (5.6kDa) to contain cathepsin D in β-acetate. The second site near the C-terminal region of the myloid peptide should also cleave APP Suggests not.   In fact, the precursor substrate for such C-terminal cleavage is also labeled with Mr (10.0 kDa). It was identified in band 5 shown. The size of this fragment is such that it spans the entire C-terminal region. Including most, if not all, parts of APP593-594 single-Glu- Implied by Val-cleavage.   APP695 appears to be an ideal substrate for cathepsin D cleavage, but It contains a number of other peptide bonds that were not cleaved by Psin D. They are The fact that it was not hydrolyzed indicates that cathepsin D within the folded APP structure. It reflects the high degree of isolation of those sites away from those approaching. Of hydrophobic residue pairs Most would be expected to reside in the hydrophobic APP protein core. same For the same reason, the sites proved to be hydrolyzed by cathepsin D (Table 5). ) Did not always contain the optimal cathepsin D recognition motif. Will be revealed. In order to be located on the protein surface, such sites are D does not contain more polar or charge than is ideal for catalytic cleavage. I have to. In this regard, 3 of the 5 internal cleavage sites are cleavable It should be noted that it contained 2 proline residues in 8 residues. Such remains Groups are often associated with branch points in secondary structure or are often on the protein surface. It has to do with the turn seen.   In a side-by-side immunoblot (Figure 12), some of the peptides produced (indicated by arrows) Is a monoclonal antibody C286.8A against the N-terminal residue of β-amyloid. Reacted. They move to the same position as Band 3 (Table 5) in Figure 12a. Band, Mr 9-10 kDa doublet, M co-migrating with band 5 (Table 5) in Figure 12a. r 14 kDa doublet, 16-18 kDa doublet migrating with band 6 in Figure 12a, And a Mr 40 kDa band was included. Of the sequenced bands (Table 5), the bands Only 3, 5 and 6 were in the immunoblot of Figure 12c. Co-migrated with the more detected band. Consistent with this, those in Table 5 N-terminal sequence suitable for inclusion of β-amyloid epitope only in the same three bands And was of size.   The time course of the formation of β-amyloid immunoreactive degradation products described in FIG. At different molar ratios of cathepsin D and APP in the presence and absence of pepstatin A It was performed both in the presence (Figure 13). In the absence of inhibitor, each initially had about Mr. A time-dependent accumulation of low molecular weight fragments starting from the formation of 16-18 and 28 kDa bands. Was observed. At 2 hours, a band of Mr about 40 kDa was observed. 16-18kDa and 40kD The band of a was further enhanced after 2 hours, but the intensity of the 28 kDa band was It remained constant over time. The intensity of 16-18kDa and 40kDa is less than 8 hours later Did not increase above. Between 8 and 21 hours, Mr at about 14, 10 and 5.6 kDa The intensity of the detectable band increased substantially. The latter three bands are 16 ~ It did not enhance in parallel with the 18 or 40 kDa band, so it was a 14, 10 and 5.6 kDa band. Is probably the result of secondary degradation of any or all of the 16-18 kDa or 40 kDa bands. I wondered. The 16-18, 10 and 5.6 kDa bands described in Figure 13 are listed in Table 5. And corresponds to the same Mr band as shown in Figure 12c. Observed in Figure 13 All bands were inhibited by pepstatin A, which means that they Confirm that this was caused by the action of D.   FIG. 23 shows the shape of cathepsin D, including those identified by the time Table 5 was generated. It provides up-to-date information on the sequences of the APP fragments that are made. It also converts the sequence to Figure 12c. Correlate with the particular C286.8A immunoreactive band observed in. Each C286.8A immune reaction For the sex band, the corresponding segment of the sequencing blot is C286.8A epi Includes a taupe and is therefore of sufficient size and size to account for the immunoblot band. One or more fragments with rows were given (Fig. 23a). Other than N-terminal of mature βAPP In addition, digestion with CD results from 7 different cleavages at the N-terminus of βAPP (Fig. 23b). And the reported specificity of cathepsin D (A. Moriyama et al., 1990, J. et al. Biochem., 88: 619; van Noort et al., 1989, J. Biol. Chem., 264: 14159; and And M. Tanji et al., 1991, Biochem. Biophys. Res. Comm., 176: 798) Match. The fragmentation pattern (Figure 23b) shows that the formation of the 5.5kDa fragment was a 38kDa peptide. Or the N- and C-termini of large precursors such as the unidentified transient 28 kDa fragment of Figure 13 It suggests that it is caused by nibbling. 5.5kDa exempt Epidemiologically reactive fragments appear to derive directly from 10-12 kDa fragments with the same sequence. That And the Mr 15-16 kDa and 18-19 kDa fragments show full-length copies of βAP. Appears to be of sufficient size to include. Apparently, 10-12kDa immune response Other products, such as those resulting from further processing of the responsive fragment, are likely to No longer detected due to decomposition or loss during electroblotting Maybe.   Catheps as a major protease in amyloid formation in Alzheimer's disease The close relationship of Shin D now accounts for other observations made regarding this disease . First, APP accumulates in lysosomes and is processed there to amyloid. There is a lot of evidence that it gives rise to forming fragments (Haas et al., 1992, Nature, 357: 500). ). Lysosomal acidic pH favors amyloid deposition (Burdick et al., 1992) , J. et al. Biol. Chem., 267: 546). Second, cathepsin D is a lysosomal protease While at the same time, there is considerable level associated with amyloid deposits in Alzheimer's brain. Has also been demonstrated by histochemistry (Cataldo et al., 1990, Proc. N. atl. Acad. Scl. USA 87: 3861).   Third, β-amyloid released by cells in culture is β-amyloid 1- 3 amino acids N-terminal to the abundant sequence beginning with the Asp 597 residue commonly found in 42 Contain a small amount of N-terminal sequence beginning at a residue Val 594 (Haas et al., 1992, Natur. e, 359: 322). This small amount of sequence is probably at position 593-594, ie cathepsin D. The same site that has just been proven to undergo specific proteolysis by It is caused by the direct proteolysis of the Glu-Val bond of E. Cathepsin D is -Glu- Since both Val-bonds and -Met-Asp-bonds can be hydrolyzed, Haas et al. Specificity required to form both two sequenced β-amyloid fragments It is emphasized here to have.   Fourth, the cysteine protease inhibitor E-64 and leupeptin are LC-99 cells. Had no effect on β-amyloid release by vesicles Inhibitors suppressed this release (Shoji et al., 1992, Science, 258: 126). Β-amyloid formation by these cells was It is shown that it was catalyzed by the enzyme. The remaining proteases that carry out such reactions Candidates should not be blocked by the cysteine protease inhibitors used in those studies. It may be lysosomal cathepsin D.   Furthermore, APP has a series of hydrophobic residues between the C-terminus of β-amyloid and the membrane anchor sequence. Contains residues. Some peptide bonds in this region are hydrolyzed by cathepsin D. Can be decomposed. In fact, the -Leu-Val-peptide bond at positions 645-646 is a PEPTIDE SORT computer. It is emphasized that it is a cathepsin D recognition site that is possible by the program. this The site may segregate with certain forms of familial Alzheimer's disease (FAD) Close to the positions of the three point mutations shown. Cleavage within this region and Cleavage of the -Glu-Val- bond at positions 593-594 may explain the size of band 3 in Table 5. Can be. The FAD mutation at this site is due to cathepsin D AP within this region. It will increase the rate of P cleavage.   Probably -Asn-Leu- to -Glu-Val-cleavable linkages at S2 'and S3' sites. Mutations in the sequence may increase cathepsin D cleavage. Whether this is the case To investigate, we use the substrate N-dansyl-Ile-Ser-Glu-Val-Lys-Met-As. p-Ala-Glu-Phe-Arg-NH₂(SEQ ID NO: 7) and the K-M pair have been replaced by NL Thus, cathepsins that hydrolyze similar peptides that mimic the FAD mutations described above. The abilities of D and P2 enzyme peak B were compared (Fig. 14). Both enzymes are M- in the long-term time frame The wild type peptide was cleaved at the D and E-V bonds, while a short incubator was used. Only a few cleavages were observed at the basation time (Fig. 14). In contrast , The cleavage of the mutant peptide by both enzymes is more than that observed with the wild type peptide. It happened at an initial speed of 30 to 50 times. The single metabolite it produces holds for 4.4 minutes. It has a longevity and was not previously seen using wild type peptides. The product was then purified and mass analyzed [M-H]⁺= 907 and amino acid composition analysis Was identified as N-dansyl-ISEVNL. Therefore, this product is the L-D bond of the substrate. Must result from hydrolysis during. This cleavage by cathepsin D is β- It also releases peptides with the same N-terminus as found in the predominant form of amyloid, and The -NL-mutation found in the specific early-onset FAD of the lever is an amyloidogenic protease By providing a site that is more rapidly cleaved by cathepsin D, β-amido It provides a mechanism to increase the rate of loid formation. Possible β -Increased rate of amyloid accumulation is due to the early-onset form of Alz associated with this APP mutation. It can induce Heimer's disease.   Cathepsin D-induced wild type holo βAPP695 and βAPP695ΔNL (the latter is The rates of hydrolysis of 595L596D to NL with the FAD mutation were compared (Figure twenty four). At pH 5.0, both substrates were hydrolyzed at comparable rates with the 18-19 kDa fragment and 15-16 kDa. A fragment was generated. In contrast, a 10-12 kDa fragment formed from wild-type βAPP695 And the 5.5 kDa fragment were barely detectable by 20 hours, while βAPP69 In 5ΔNL hydrolysis, fragments with their same Mr value were clearly observed. 5.5 The kDa and 10-12 kDa bands are 5-1 less than the corresponding rates in wild-type βAPP695. It was estimated to be formed from βAPP695ΔNL at a rate 0 times greater. βAPP695 The ΔNL fragment co-migrated with the wild type βAPP695 fragment as well as the recombinant C-100 fragment, The idea that these fragments result from cleavage at or near the N-terminus of βAP Match with E.   ΔNL F to increase the rate of hydrolysis catalyzed by cathepsin D The effect of the AD mutation is that the N-dane occurs at the LD bond adjacent to the N-terminal residue of βAP. Greatly increases the rate of CD-catalyzed hydrolysis of sil-βAPP591-601-amide Consistent with the effect of the same substitution (FIG. 14), and 5.5 kDa and 1 from βAPP695ΔNL. Increased rate of formation of the 0 kDa fragment with either 593E-594V or 596M-597D binding This is because cleavage of 596L-597D is easier than that of wild-type βAPP of Escherichia coli. Suggest that. The βAPP695ΔNL fragment is presumed to contain the full-length copy of βAP. And, as such, is characteristic of the Swedish form of familial Alzheimer's disease. Can act as an intermediate for id deposition. Cathepsin D-dependent amilo of APP The effect of the Swedish mutation on increasing the rate of idation processing was confirmed by the Further emphasize the importance of CD in id formation.   An important candidate for amyloidogenic proteases in Alzheimer's disease And the identification of cathepsin D in the past made efforts to develop inhibitors for the treatment of this disease. Greatly help. For example, specific cathepsin D inhibitors are toxic to β-amyloid. Inhibiting sexual accumulation could provide therapeutic benefit. In this specification The new information provided to us is about other asparagus, such as renin and HIV protease. As demonstrated in the design of new inhibitors of formate proteases, It makes the theoretical design of binding inhibitors relatively easy.   Alternatively, cathepsin D is now randomized or semi-randomized in the drug library. High-performance using an in vitro peptide assay to identify therapeutic inhibitors throughout the study Suitable for use in throughput screening. Appropriate for such purpose As a sei, the N-dansyl peptide described in Examples 2 and 3 of the present invention is used. I can give you a essay.Example 10. Identification of serine proteases with C-terminal APP processing specificity   Table 4 shows that the human brain is capable of performing C-terminal processing of recombinant APP. , And in some cases their serine protea It was shown that the enzyme could be inhibited by aprotinin. Such protea In order to attempt easier isolation of the protease, aprotinin-cephalo was used as an initial step. Another isolation scheme has been devised that incorporates affinity purification on Example 1, method 2).   Application of this procedure to further purification of the P2 fraction was successful in isolating APP degrading activity. Was (Fig. 15). Recovered from the aprotinin-sepharose column by acid elution. The resulting active fraction was further purified on a Mono-Q column (Fig. 16). The active fraction (Fig. 16a) , To the C terminus of APP When analyzed by immunoblotting using a polyclonal antibody, it was found to be 11 kDa and 14 kDa. And demonstrated the ability to form a 18 kDa C-terminal APP fragment (Fig. 16b). Minimum product Migrated with the recombinant C-100 standard. Anti-β-amyloid monoclonal antibody C Re-assay for APP degrading activity in the active fraction using 286.8A showed the same three products. Band detection was reached (Fig. 16b). C286.8A antibody as described in Example 6 (iii). Recognizes the first seven amino acid residues of the β-amyloid peptide. Studies show that all three products contained full length β-amyloid.   One or more of those product peptides are amyloid or β-amido. By further processing of those peptides at the C-terminus of the Lloyd region, β-amido Lloyds can be generated. Therefore, the serine involved in the formation of those products Protease activity may play some role in amyloid formation.   Enzyme activity forming the above 11, 14 and 18 kDa product bands is broad from Mono-Q Elutes as a peak of, and probably results from the action of multiple proteases. Yes. Based on the recovery of the A280nm absorbing component from the Mono-Q column, pool X, Y and Three proteolytic activity pools were prepared from the Mono-Q column fractions designated Z and Z. (Example 1, Method 2).   It is not possible to rule out protein aggregation that can occur during chromatography However, chromatographs of each pool on Superdex 75 consistent with an apparent Mr> 75kDa Enzyme activity was recovered in the interstitial volume in the ruffee (data not shown). Pool Y represents the purest pool when analyzed on SDS-PAGE and is of the order of 100 kDa. A major staining band was shown at Mr. Select pool Y for further characterization I chose. The pH dependence of APP hydrolysis by pool Y is pH 7. Optimal values were shown between 9 (Fig. 17a), and the enzyme activity showed a sodium chloride concentration of 42 mM. It was gradually inhibited by increasing over (Fig. 17b). Sense of inhibitor of the enzyme Passivity studies (Figure 18a) show that it is inhibited by PMSF and aprotinin It is a serine protease because it is not affected by tatin A, E-64 or EDTA. This was confirmed (Fig. 18a). Benzamidine, a serine protease inhibitor Had no effect on this enzyme, which means that the trypsin-like endoprotea Suggest that it is not a case. Probably the enzyme is at the S1 subsite Chymotrypsin-Phase with Specificity for Cleavage of Substrates Containing Neutral Hydrophobic Residues Millie's.   Therefore, further inhibitor studies (Figure 18b) showed that the activity of pool Y protease It is strongly inhibited by trypsin inhibitor II, α-2-antiplasmin and TPCK. Was shown. Weak inhibition by chymostatin and α-1-antichymotrypsin was also observed. However, TLCK did not inhibit at all. Cathepsin G plays a role in APP processing Has been proposed by other researchers to achieve the Immunoblot analysis of pooled Y protease fraction using internal antibody The presence of phosphoprotease could not be detected. 11, 14 and 18kDa N-terminal sequence analysis will identify the cleavage site, which is already underway.Embodiment 11 FIG. Design of therapeutic cathepsin D inhibitors   This example is described in Schechter et al., 1967, Biochem. Biophys. Res. Use the nomenclature of Commn., 27: 157. In this nomenclature, The amino acid numbers in the adjacent substrates correspond to the peptide bonds that are cleaved by the enzyme. Turn according to their position It is numbered. The peptide substrate amino acid side chain on the N-terminal side of the scissile bond is P1 to Pn are numbered consecutively as the distance from the sexual bond increases. Off The peptide substrate amino acid side chains on the C-terminal side of the scissile bond are sequentially numbered P1 'to Pn'. It is numbered. The P1 and P1 ′ amino acid side chains are linked to the peptide bond that is Corresponds to the amino acids involved in the formation of bonds. P1 to Pn and P1 'to Pn' The nooic acid side chain and its corresponding series of enzyme subsites S1-Sn and S1'-Sn '. It is imagined that each forms a specific interaction. S subsidy corresponding to P side chain Interacts with the binding energy for stabilization of the protease-substrate complex. Contribute and thus add specificity to the interaction.   The approach taken in the development of peptidomimetic inhibitors has been shown to work with purified cathepsin D. The N-dansyl peptide substrate of Examples 2 and 3 was used to perform the same enzymatic assay. Either the assay or the holoAPP degradation assay described in Example 8 can be used. .Optimal peptide length for proteolytic cleavage by cathepsin D in vitro Array quantification . Sequence Dns-Ile-Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg-NH₂(Distribution Apparent kinetics of enzymatic hydrolysis, starting with the dodecapeptide in column no. 7) Of the peptide from either the N-terminus or the C-terminus for biological parameters The effect of shrinkage is measured at acidic pH and optimum ionic strength. Optimal length peptide Of changes in amino acids at each position in Km and Vmax of hydrolysis Measure the sound.Inhibitor synthesis . Suitable for the essential amino acid sequence (from 1a and b above) required for optimal cleavage A peptidomimetic compound containing the appropriate spacer is synthesized. In those peptides Has the same amino acid sequence as found near the cleavage site in the APP substrate, eg Glu-Ile-Ser-Glu-Val-Lys- Met-Asp (SEQ ID NO: 4) and Trp-His-Ser-Phe-Gly-Ala-Asp-Ser (SEQ ID NO: 5) Or, alternatively, to study the ability of cathepsin D to inhibit in vitro. Based on the amino acid sequence that was found to confer optimal binding to cathepsin D based on You can choose. Glu-Ile-Ser-Glu-Val-Lys-Met-Asp (SEQ ID NO: 4) and And Trp-His-Ser-Phe-Gly-Ala-Asp-Ser (SEQ ID NO: 5), a P1-P1 'bond Are EV and FG, respectively.   The above sequences or sequences showing optimal cathepsin D inhibition (probably N and / or (Including short variants with C substitutions) to obtain any possible stereochemical configuration. Using the appropriate synthetic route and to any of the following standard spacer groups: Thus the -CO-NH- atom of the peptide bond between P1 and P1 'has been replaced, Synthesize peptide inhibitors: reduced amides, hydroxy isosteres, ketone isosteres, Dihydroxy isostere, statin analog, phosphonate or phosphonamide, reverse Inverted amide. Most of those inhibitors will function as transition state analogs.   In vitro assay of the invention (N-dansyl peptide assay or holo APP Of those first generation compounds as measured using any of the degradation assays) Efficacy can be optimized by some or all of the following:   (I) Addition or deletion of adjacent amino acid residues;   (Ii) Alteration of amino acid side chain morphology (D or L) at each position of the inhibitor;   (Iii) Boc or acetyl (N-terminal) or O-Me, O-benzyl or Substitution of N- and C-termini with protecting groups such as N-benzyl (C-termini).   Besides the inhibitors theoretically developed according to the above method, other known cathepsin D Inhibitors can also be used as inhibitors for the treatment of Alzheimer's disease or with optimization of inhibitory activity and Overall as a starting point for the development of new derivatives for the treatment of Luzheimer's disease. Or partially used. Examples of such inhibitors include: 1-deoxynojirimidine (Lemansky et al., 1984, J. Biol. Chem., 259: 10129); diazoacetyl-norleucine methyl ester (Keilova et al., 1970, Fe bsLett., 9: 348); Gly-Glu-Gly-Phe-Leu-Gly-Asp-Phe-Leu (SEQ ID NO: 6) (Gub enseck et al., 1976, Febs Lett., 71:42); pepsin inhibitor from Ascaris (Keilova). 1972, Biochem. Biophys. Acta, 284: 461); Pepstatin (Yamamoto et al., 1 978, European Journal of Biochemistry, 92: 499).Example 12. C-terminal by human embryonic kidney (HEK) 293 cells maintained in culture Effect of cathepsin D inhibitor pepstatin A on the formation of terminal APP fragments   In this example, peptatin A, an inhibitor of cathepsin D activity, was tested in vitro. The same size as shown to be formed from APP 695 by cathepsin D in HE producing a 15-kDa C-terminal APP fragment and releasing it into tissue culture medium It is shown to inhibit the ability of K293 cells in vitro. HEK cells transfect Release β-amyloid from the cleaved APP 695 and thus amyloidogenic A It is known to contain a protease required for PP processing [C. Haa s et al., 1992, Nature, 359: 322]. Therefore, those cells should be studied for β-amyloid formation. It provides a suitable cell model for research. APP751 / present in those cells The endogenous level of 770 served as a substrate for the studies outlined below. We are 4 00 ml HEK293 cells were grown in suspension culture. Some cultures used DMSO solvent only (maximum Final concentration 0.01% v / v) or DMSO + 10 μM final concentration of pepstatin A Cells were grown in containing medium. As is clear from Figure 19a, DMSO also DMSO + pepstatin also increased the growth rate of HEK293 cells at the concentrations used. It had no adverse effect. DMSO treated cells or DMSO + pepstatin A treated cells An aliquot (185 ml) of medium taken in late log phase from either is recommended Procedure [Axen R. Et al., 1967, Nature, 214: 1302] by CNBr-activated Sepharose 4 It was immobilized on Sepharose 4B using B (Pharmacia). Monoclonal C28 It was passed through a column of the same size (1.6 x 5 cm) of 6.8A (Example 6). 500m before sample loading The column was equilibrated with 100 mM sodium hydrogen carbonate buffer pH 8.3 containing M NaCl. HE The β-amyloid-containing APP fragment released into tissue culture medium by K293 cells is Bound to immobilized monoclonal antibody, then containing 0.025% v / v Triton X-100 1 The column was eluted at a flow rate of 1 ml / min by washing with 00 mM glycine pH 2.4. Was. The elution fraction (4 ml) may be bound to the column and then eluted from the column. The immunoblot method of Example 8 was used to detect any APP fragment. I did. Immunoblot detection was performed using the anti-C-terminal antibody of method i) of Example 6. Was. The fraction detected by this method is the N-terminal heptapeptide of β-amyloid. Sequence (to illustrate binding to immobilized C286.6A) and C-terminal region or one of its Would have to contain a moiety (to account for reactivity with anti-C-terminal antibody) U.   Figure 19b shows growth in the presence of either DMSO alone or DMSO + pepstatin A. Recovered in the eluate fraction from the column of immobilized C286.8A loaded with cultivated cell medium. The amount of C-terminal fragment is compared. Chromat The graphs were performed in parallel under the same conditions. Obviously, Pepstatin A Treatment with the eluted 15-16 kDa can be detected by immunoblot Significantly reduced the amount of APP-derived fragments. This fragment has the N-terminal sequence G-A-D-S-V- Fragments formed in vitro by cathepsin D with P-A- (Table 5 and Figure 23) Small size with the N-terminus corresponding to some forms of β-amyloid It may represent an intermediate during cell formation of the 5.6 kDa fragment. Cathepsin D in vitro No other APP fragment formed was detected in this experiment. Undetected fragments are detected It is present at levels below the threshold or is modified intracellularly by other proteases. It may have been decomposed into. This experiment characterizes the formation of amyloid in cells HEK293 cells, a generally accepted cell line for At least one APP fragment that more closely resembles the APP695 fragment formed in vitro. Generating and releasing, and the formation of this fragment results in a non-toxic amount of cathepsin D inhibitor. Prove that it will be more hindered. Thus, peptide-based inhibitors of cathepsin D Have utility in altering the APP processing of cells.Example 13. Inhibition studies   N-dansyl-ISEVKMDAEFR-NH with cathepsin D₂Using in vitro hydrolysis of 250 selected from the renin and HIV-protease inhibitor programs Of peptidic compounds for their ability to inhibit human cathepsin D. I learned. Among them, the compounds identified in Table 6 below are effective cathepsics. It showed an inhibitory activity on protein D.   The structures of the inhibitors corresponding to the numbers given in Table 6 are given in Table 7 below:   In all the above formulas, the abbreviation "BOC" represents tert-butoxycarbonyl.   The above inhibitors can be prepared as follows: Inhibitors 1, 2, 3, 4, 10 and 20 correspond to US patent application Ser. No. 08 / 059,488 filed May 10, 1993 Described in German patent application DE 4,215,874 filed on May 14, 1992 . The disclosures of both of these applications are incorporated herein by reference. Their inhibition The agents are disclosed to be antiviral agents, especially HIV protease inhibitors. Obstruction Harmful agent 1 was prepared as follows.   First stage: 614 mg (2.20 mmol) of (2R) in 10 ml of anhydrous dichloromethane -N- (tert-butoxycarbonyl) -2-amino-2- [2- (1,3-dithio) Oran-2-yl)] acetic acid (EP 412 350) and 337 mg (2.20 mmol) 1-hydr 434 mg (2.10 ml) of a stirred solution of roxybenzotriazole (HOBT) cooled to 0 ° C. Limol) dicyclohexylcarbodiimide (DCC), and the mixture Stir for a minute. Then 1.10 g (2.20 mmol) of 1- in 10 ml of dichloromethane {(2R, S, 4S, 5S)-[5-amino-6-cyclohexyl-4-hydro Xy-2- (1-methyl) ethyl-hexanoyl]}-S-isoleucinyl-2 -Pyridylmethylamide dihydrochloride (EP 437 729) and 0.88 ml (8.0 mmol) N -Add a solution of methylmorpholine dropwise. Remove the cooling bath and allow the reaction mixture to Stir for 2 hours at warm temperature. The end of the reaction is determined by thin layer chromatography. The resulting urea was removed by filtration, the filtrate was concentrated in vacuo and 90 g of silica gel was added. Crude by chromatography on column (dichloromethane: methanol 95: 5) Purify the product. 1.29 g (88% of theory) of compound: Is obtained as a pale powder.   Second stage: 2.41 g (3.28 m) in 17 ml of 4N gaseous hydrogen chloride / anhydrous dioxane solution A solution of said compound 27 in (mol) is stirred at 0 ° C. for 30 minutes. Then 15 ml of toluene Is added and the mixture is concentrated in vacuo. This process is repeated two more times, then the residue Is triturated with ether, suction filtered and dried under high vacuum on potassium hydroxide (KOH). And 2.29 g (98% of theory) of compound: Is obtained as a colorless powder.   Third stage: 0.80 g (2.40 mmol) of (2S) in 20 ml of anhydrous dichloromethane -3-tert-Butylsulfonyl-2- (1-naphthylmethyl) propionic acid [H . Buhlmayer et al., 1988, J. Med. Chem., 31: 1839] and 0.40 g (2.64 A stirred solution of rimol) HOBT cooled to 0 ° C. was treated with 0.52 g (2.52 mmol) DCC. And then Stir for 5 minutes. Then before 1.55 g (2.19 mmol) in 30 ml dichloromethane A solution of Compound 28 and 0.96 ml (8.74 mmol) of N-methylmorpholine was added dropwise. Then, the reaction solution is stirred at room temperature for 2 hours. The resulting urea is removed by filtration and the filtrate Concentrate in air and optionally chromatograph on 360 g silica gel ( Purify the crude product with dichloromethane: methanol 95: 5). Colorless powder 586 mg (28% of theory) of non-polar (2R) -isomer and 690 mg (33 %) Of the polar (2S) -isomer is obtained.   Inhibitors 2,3,4,10 and 20 are suitable acids with suitable amine hydrochlorides (of which The starting materials are known or can be prepared by conventional means) and cups It can be similarly prepared by ringing. In the case of inhibitors 3 and 20, It may be necessary to start with the compound having the formula: The preparation of compound 29 is similar to that of compound 27, except that 249 mg (0.89 mmol ) Of (2R) -N- (tert-butoxycarbonyl) -2-amino-2- [2- ( 1,3-dithiolan-2-yl)] acetic acid [EP 412 350] and 440 mg (0.81 mmol) ) 1-{(2R, S, 4S, 5S)-[5-amino-6-cyclohexyl-4 -Hydroxy-2- (2-propenyl) hexanoyl]}-S-isoleucine Starting from lu-2-pyridylmethylamide dihydrochloride [prepared according to EP 437 729] , And chromatography on 24 g of silica gel (dichloromethane: methano 9: 1) to purify the crude product. 553mg (93%) of compound as pale powder Item 29 is obtained. Then the preparation of the amine hydrochloride is similar to that of compound 28, but However, starting from 560 mg (0.91 mmol) of compound 29. 452 mg as colorless powder ( 91%) of the amine hydrochloride is obtained.   Inhibitor 10 would require a starting material of the formula: The preparation of compound 30 is similar to that of compound 27, except that 258 mg (0.92 mmol ) Of (2R) -N- (tert-butoxycarbonyl) -2-amino-2- [2- ( 1,3-dithiolan-2-yl)] acetic acid [EP 412 350] and 500 mg (0.84 mmol ) 1-{(2R, S, 4S, 5S)-[5-amino-6-cyclohexyl-4 -Hydroxy-2- (2-phenyl) hexanoyl]}-S-isoleucinyl- Starting from 2-pyridylmethylamide dihydrochloride [prepared according to EP 437 729], Chromatography on 20 g of silica gel (dichloromethane: methanol   95: 5) to purify the crude product. 593 mg (90%) of compound 3 as an amorphous powder You get 0. Then the preparation of the amine hydrochloride is similar to that of compound 28 but only 589mg Starting from (0.75 mmol) of compound 29. 484 mg amine hydrochloride as a colorless powder A salt is obtained.   Inhibitor 7 may be European Application Publication EP 0 472 077 published February 26, 1992. Are known and their entire contents are incorporated herein by reference. The inhibitor Are disclosed therein as inhibitors of HIV protease activity.   Inhibitors 8 and 13 are described in European patent application EP 0 441 9 published Aug. 14, 1991. Known from 12, the entire content of which is incorporated herein by reference. The obstacle Harmful agents are disclosed therein as inhibitors of renin.   Inhibitors 9, 15, 16 and 19 are European patent applications published on 26 February 1992 Open EP 0 472078 (this is the same as US Pat. No. 5,147,865 issued Sep. 15, 1992) , And the entire contents of both applications are incorporated herein by reference. You. The inhibitor is disclosed therein as an inhibitor of HIV protease activity .   Inhibitors 11 and 12 are described in pending US Application No. 07 / 920,216 filed Jul. 24, 1992. And European patent application EP 0 528 242, published February 24, 1993, 8 August 1991. It is described in the German patent application DE 41 26 485 filed 10 / month. Those 3 applications Is incorporated herein by reference. The inhibitor is It is disclosed as an inhibitor of Rotase activity. Both of these inhibitors are It can be prepared as:   First stage: 5.00 g (20 g in 100 ml of a 4N solution of gaseous hydrogen chloride in anhydrous dioxane) .21 mmol of (S) -2- (tert-butoxycarbonylamino-1-phenyl) Lubut-3-ene [J.R. Luly et al., 1987, J. Org. Chem., 52: 1487] Stir at warm temperature for 30 minutes. Then 15 ml of toluene are added and the mixture is concentrated in vacuo. . This work The procedure is repeated two more times, then the residue is triturated with a little ether, suction filtered and KO 3.69 g (99% of theory) of the compound when dried under high vacuum on H: Is obtained as colorless crystals.   Second stage: 4.81 g (22.13 mmol) of N- (te in 40 ml of anhydrous dichloromethane. rt-Butoxycarbonyl) -L-valine and 3.29 g (24.35 mmol) HOBT at 0 ° C. The stirred solution is treated with 5.29 g (25.65 mmol) DCC and stirred for 5 minutes. Next Then 3.70 g (20.12 mmol) of compound 31 and 8.85 g (80. A solution of 48 mmol) N-methylmorpholine is added dropwise. Remove the cooling bath The reaction mixture is stirred at room temperature for 2 hours. Finish the reaction by thin layer chromatography Therefore, it is determined. The resulting urea was removed by filtration and the filtrate was concentrated in vacuo and Chromatography on 450 g of silica gel (dichloromethane: methanol 95: 5) to purify the crude product. 6.07 g (87% of theory) of compound: Is obtained as a colorless foam.   Third stage: 6.08 g (17.53 g) in 100 ml of 4N gaseous hydrogen chloride / anhydrous dioxane solution (32 mmol) of the compound 32 is stirred at room temperature for 30 minutes. Then 15 ml of toluene Add and concentrate the mixture in vacuo. This process is repeated two more times and then the residue is Triturate with a small amount of ether, filter with suction, and dry under high vacuum on KOH to give 4.90 g (theoretical 99% of value) compound: Is obtained as a colorless powder.   Fourth stage: 1.50 g (4.47 mmol) of (2S) in 15 ml of anhydrous dichloromethane -3-tert-butylsulfonyl-2- (1-naphthylme 1839] and 0.66 g (4.92 mmol) of HOBT at 0 ° C. with stirring at 0.97 g (4.69 mmol). DCC and stir for 5 minutes. Then 1.15 g in 10 ml of dichloromethane (4.07 mmol) of compound 33 and 1.80 ml (16.27 mmol) of N-methylmorpholine Solution is added dropwise and the reaction is stirred at room temperature for 1 hour. The resulting urea is filtered The filtrate was concentrated in vacuo and chromatographed on 270 g of silica gel. Purify the crude product by means of a charge (dichloromethane: methanol 95: 5). 2.01 g (88% of theory) of compound: Is obtained as a colorless foam.   Fifth stage: A 0 ° C. stirred suspension of compound 34 in dichloromethane was added with 2 equivalents of m-chloro. Treat with perbenzoic acid (MCPBA) (80% strength) and stir at this temperature for 2 hours. Next Then another 1 equivalent of MCPBA is added and the mixture is stirred for another hour at room temperature. Then Ethyl acetate was added and the reaction mixture was adjusted to 10% Na₂SO_ThreeStir in solution. Separate the organic phase , NaHCO_ThreeWash 3 times with solution and MgSO_FourDry on top. The solvent is evaporated in vacuum The residue was triturated with a little ether / pentane and the compound: Is obtained as a colorless powder.   6th stage: Compound 35 and (2S) -2- (trifluorome in n-propanol Til) pyrrolidine [G.V. Shustov et al., 1987, Isvest. Akad. Nauk. SSSR, 1422 ( engl)]] (for preparing inhibitor 11) or (2S) -2- (trifluoro) Methyl) piperidine (inhibitor Stir any of the solutions in 13) at elevated temperature in a pressure vessel. Cooled After that, the reaction mixture is concentrated in vacuo and chromatographed on silica gel. To purify. The inhibitor is obtained after trituration with n-pentane.   Inhibitors 5 and 6 are within the general teaching of EP 0 441 912 (supra), among which It can be prepared according to the preparation method taught. For example, inhibitor 5 is Can be prepared as follows:   First stage: 300 g (1.91 mol) of L-phenylalanine and 360 ml of dioxane and 3 60 ml H₂Suspend in O. 432.9 g (1.98 mol) of di-tert- with stirring at pH 9.8 Add butyl dicarbonate. Keep the pH constant with about 975 ml of 4N NaOH . After 16 hours, the reaction mixture was extracted with ether and the aqueous phase adjusted to pH 3-4 with citric acid. , Then extracted twice with ether and twice with ethyl acetate. Combine the organic phases and water And wash three times. Concentrate in a rotary evaporator and wash with diethyl ether / hexane. Crystallized from xane, 291.6 g (60.7%) of compound: Is obtained.   Second stage: 265 g (1.0 mol) of compound 36 was dissolved in 2 l of methanol, and 20 g of 5 Hydrogenate on% Rh / C at 40 atmospheres for 5 hours. Touch by suction filtration through Celite The medium is removed, the catalyst is washed with methanol and the resulting solution is concentrated. 271g (100%) compound: Is obtained.   Third stage: 163.0 g (0.601 mol) of compound 37 and 40.3 g (0.661 mol) of N, O- Dimethylhydroxylamine is dissolved in 2 l methylene chloride at room temperature. At 0 ° C Then, 303.5 g (3.005 mol) of triethylamine is added dropwise (pH ~ 8). Most At high -10 ° C, add 390.65 ml (0.601 mol) of 50% solution of n-PPa in methylene chloride Add dropwise. The mixture is warmed to 25 ° C overnight and stirred for 16 hours. Then concentrate the reaction mixture Then add 500 ml of saturated bicarbonate solution to the residue and stir the mixture at 25 ° C for 20 minutes. . After extracting 3 times with ethyl acetate, the organic phase is washed with Na₂SO_FourDry on and concentrate. Crude yield: 178 g (94.6%). Chromatography of the crude product on silica gel Phase CH₂Cl₂: CH_ThreePurify with OH 98: 2). 136.6 g of compound: Is obtained.   Fourth stage: 63.7 g (0.21 mol) of compound 38 in a flame dryer under nitrogen Was dissolved in 1.5 l of alumina-treated ether and 10 g (0.263 mol) at 0 ° C. LiAlH_FourIn portions and then the mixture Stir for 20 minutes at 0 ° C. Then 1 l of H₂50 g (0.367 mol) KHSO in O_FourSolution of Carefully add dropwise at 0 ° C. The phases are separated and the aqueous phase is combined with 3 x 300 ml diethyl ether. And the combined organic phases are extracted 3 times with 3N HCl and NaHCO 3._Three3 times with solution and then with NaCl Wash twice with liquid. The organic phase is Na₂SO_FourDry on and concentrate. 45g (84.1%) conversion Compound: Is collected. Compound 39 is immediately further processed or stored at -24 ° C for 1-2 days You.   Fifth stage: 14.6g (35mmol) of "instant ylide" (Fluka 69500) is suspended in 90 ml of anhydrous tetrahydrofuran. Cooling in ice While and at a reaction temperature of 20-25 ° C, in 45 ml of anhydrous tetrahydrofuran. A solution of 9.0 g (35 mmol) of compound 39 is added dropwise. Stir the reaction mixture for 15 minutes And then poured into 250 ml of ice, 150 ml of ethyl acetate / n-hexane (3: 1) each time. Extract twice with. Na₂SO_FourAfter drying over and concentrating, the residue is dried over silica gel. Purify by chromatography (mobile phase ether: hexane 7: 3). 3.2g ( 40.0%) of the compound: Is obtained.   6th stage: 202.4 g (0.8 mol) of compound 40 is dissolved in 1000 ml of mesitylene, Heat to 140 ° C with a water trap. At this temperature, 197 g in 800 ml mesitylene (1.6 mol) of N-benzylhydroxylamine and 1.6 mol of acetaldehyde The mixture is added dropwise over 2 hours. After 4 and 8 hours reaction time, equal volumes of Add N-benzylhydroxylamine and ethyl aldehyde in styrene dropwise. You. After a total reaction time of 16 hours, the mixture was concentrated and diethyl ether was added to the residue. Then mix the mixture with 1M KHSO_FourWash with solution. Na₂SO_FourDried over and concentrated After that, the residue is chromatographed on silica gel (mobile phase ether: hexane 3 : 7). Compound: Is obtained.   7th stage: 18.1 g (45 mmol) of compound 41 (diastereomer C) in 300 ml Dissolve in methanol. After addition of 14.2 g (225 mmol) ammonium formate, the device Thoroughly N₂Flush and add 3.6 g palladium / carbon (10%) You. The mixture is stirred at reflux for 3 hours. After cooling, remove the catalyst by filtration. Dissolved, concentrated the solution, dissolved the residue in ethyl acetate, and saturated bicarbonate solution. And wash twice. The organic phase is dried over sodium sulfate and filtered Dried, concentrated and dried under high vacuum. 11.36 g of compound: Is obtained.   Eighth stage: 6.6 g (21 mmol) of compound 42 is dissolved in 500 ml of methylene chloride. Water exclusion (CaCl₂Tube) while stirring in pentamethylene anhydride in methylene chloride [50 ml salt 2.16 g (21 mmol) of pentanoic acid and 2.16 g (10.5 mmol) of dicarboxylic acid in methylene chloride Was prepared from cyclohexylcarbodiimide] at room temperature. 3 hours Afterwards, concentrate, take up the residue in ethyl acetate, wash with saturated bicarbonate solution, and Dry over sodium sulfate. Filtration and concentration followed by drying under high vacuum. 8.0g ( 95.2% of theory) of compound: Is obtained.   9th stage: 7.57 g (19 mmol) of compound 43 while removing water, 70 ml of 4N Stir for 30 minutes in hydrochloric acid / dioxane. The reaction mixture is concentrated and mixed with diethyl ether. Combine and evaporate to dryness. After drying under high vacuum, 5.54 g (16.5 mmol) corresponding Hydrochloride, 4.46 g (33 Limol) HOBT and 16.5 mmol Boc-Val-OH in 500 ml methylene chloride. Lend. After cooling to 0 ° C, the pH was adjusted to 8.5 with N-methylmorpholine and then adjusted to 3.5 7 g (17.3 mmol) dicyclohexylcarbodiimide are added. 16 hours at 20 ℃ After that, the urea was removed by filtration, the filtrate was concentrated, the residue was taken up in ethyl acetate and saturated. Wash with bicarbonate solution. After drying over sodium acetate, concentrate and Dry in the air. Compound: Is obtained.   Tenth stage: 1.8 mmol of compound 44 in 11 ml of 4N hydrochloric acid / dioxane for 30 minutes Stir. The reaction is concentrated, mixed with diethyl ether and evaporated to dryness. High truth After drying in air, 1.8 mmol of the resulting hydrochloride salt was dissolved in 50 ml of methylene chloride. Then cool to 0 ° C. After adding 1.8 mmol Boc-phenylalanine, The pH was adjusted to about 8 with lyethylamine and 875.2 mg (1.98 mmol) benzo Triazolyloxy-tris (dimethylamino) phosphonium hexafluorophore Add sulphate. After reacting for 16 hours at room temperature, the reaction mixture was concentrated and the residue was vinegared. Take up in ethyl acidate and wash 3 times with saturated bicarbonate solution. Inhibitor 5 in crude form Obtained and then purified by chromatography on silica gel.   Inhibitor 6 is obtained in the same manner as Inhibitor 5, except that in the eighth step methyl chloride 3-methylpentanoic acid anhydride [50 ml of methyl chloride 21 mmol 3-methylpentanoic acid and 2.16 g (10.5 mmol) disic in ren Prepared from lohexylcarbodiimide and filtered] is used.   Inhibitor 17 is within the general teaching of EP 0 472 077 (supra) and teaches therein. It can be prepared according to the prepared preparation method. For example, inhibitor 17 Can be prepared:   First stage: 5.07 g (20.00 in 100 ml of 4N gaseous hydrogen chloride / anhydrous dioxane solution) Mmol) of (S) -2- (tert-butoxycarbonylamino) -1-cyclohexyl Xylbut-3-ene [J.R. Luly et al., 1987, J. Org. Chem., 52: 1487] solution Is stirred at room temperature for 30 minutes. Then add 15 ml of toluene and concentrate the mixture in vacuo. I do. This process is repeated twice more, then the residue is triturated with a little ether and absorbed. 3.76 g (99% of theory) of the compound after draw filtration and high vacuum drying on KOH: Is obtained as colorless crystals.   Second stage: 4.63 g (21.3 mmol) of N- (ter in 40 ml of anhydrous dichloromethane t-butoxycarbonyl) -L-valine and 3.29 g (24.35 mmol) HOBT at 0 ° C. The stirred solution is treated with 5.29 g (25.65 mmol) DCC and the mixture is stirred for 5 minutes. Then 3.60 g (19.00 mmol) of compound 45 and 8.85 ml (30 ml of dichloromethane) A solution of 80.48 mmol N-methylmorpholine is added dropwise. Take a cooling bath The reaction mixture is stirred at room temperature for 2 hours. The reaction is completed with a thin layer Determined by matography. The resulting urea was removed by filtration and the filtrate was vacuumed. Concentrated in, and chromatographed on 450 g of silica gel (dichlorometa The crude product is purified by methanol / methanol 95: 5). 4.33g (65% of theory) Compound: Is obtained as colorless crystals.   Third stage: 4.32 g (12.30 g) in 100 ml of 4N gaseous hydrogen chloride / anhydrous dioxane solution A solution of 46 mmol of compound 46 is stirred at room temperature for 30 minutes. Then 15 ml of toluene Add and concentrate the mixture in vacuo. Repeat this step two more times and then remove the residue. Triturate with a little ether, filter with suction and dry under vacuum on KOH. 3.37g (theoretical value 95% of the compounds): Is obtained as a colorless powder.   Fourth stage: 4.47 mmol Boc-L-cyclohexyl in 15 ml anhydrous dichloromethane Luamine [M.C. Khosla et al., 1972, J. Med. Chem., 15: 792] and 0.66 g (4.92 mm Mol) HOBT at 0 ° C. was treated with 0.97 g (4.69 mmol) DCC, and The mixture is stirred for 5 minutes. Then 4.07 mmol of the compound in 10 ml of dichloromethane was combined. Object 47 and 1.80 ml A solution of (16.27 mmol) N-methylmorpholine was added dropwise, and the reaction solution was cooled to room temperature. And stir for 1 hour. The urea formed was removed by filtration and the filtrate was concentrated in vacuo and Chromatography on 270 g of silica gel (dichloromethane: methanol   95: 5) to purify the crude product. Thus the compound: Is obtained.   Fifth stage: 0.60 mmol of compound 48 suspended in 3 ml of dichloromethane at 0 ° C with stirring. The liquid was divided and treated with 2 equivalents of m-chloroperbenzoic acid (80% strength) at this temperature. The mixture is stirred for 2 hours. Then another 1 equivalent of m-chloroperbenzoic acid was added, The mixture is stirred at room temperature for 1 hour. Then 10 ml of ethyl acetate was added and the reaction mixture was 20 ml of 10% Na₂SO_ThreeStir in solution. The organic phase is separated and 10 ml NaHCO_Three3 in solution Washed twice and MgSO_FourDry on top. The solvent was evaporated in vacuo and the residue was washed with a small amount. After trituration with ether / pentane, the inhibitor 17 is obtained.   Inhibitor 14 corresponds to US Pat. No. 5,145,951 issued Sep. 8, 1992. 1 It is known from European Application Publication No. 0 437 729, published 24 July 991. The entire contents of both publications are incorporated herein by reference. The inhibitor is It is disclosed as an inhibitor of HIV protease activity.   Inhibitor 18 is a currently pending US patent filed on April 28, 1992. Application No. 07 / 876,697 (This is a US patent filed May 16, 1990, now abandoned. No. 07 / 524,779 (continuation application), which was published on December 27, 1990 Known from Loppa application 0 403 828. All disclosures of those three applications are for reference Incorporated herein by reference. The inhibitor is one of the It is disclosed as an inhibitor.   Inhibitor 21 can be prepared as follows: 5 ml anhydrous dichloromethane 227 mg (0.68 mmol) of (2S) -3-tert-butylsulfonyl-2- ( 1-naphthylmethyl) propionic acid [H. 104 mg (0.68 mmol) HOBT (1-hydroxybenzotriazole) was stirred at 0 ° C. The stirred solution was treated with 134 mg (0.65 mmol) DCC (dicyclohexylcarbodiimide). Allow and stir for 5 minutes. Then 400 mg (0.62 ml) in 10 ml of dichloromethane. Limol) 1-{(2R, S, 4S, 5S) -5- (S-valinylamino) -6 -Cyclohexyl-4-hydroxy-2- (1-methyl) ethyl-hexanoyl } -S-isoleucinyl-2-pyridylmethylamide dichloride and 0.27 ml (2.47 Solution of N-methylmorpholine) is added dropwise and the reaction is allowed to stand at room temperature. Stir for 2 hours. The resulting urea was removed by filtration and the filtrate was concentrated in vacuo and Chromatography on 60 g of silica gel (dichloromethane: methanol 9 The crude product is purified according to 5: 5). 68 mg (11% of theoretical value) with small polarity (2R ) -The isomer is obtained as a colorless powder. [Melting point: 187-189 ° C (decomposition); R_f= 0.15 (Dichloromethane: Methanol 95: 5): MS (FAB): m / z = 890 (M + H)⁺. ] In addition, 7 5 mg (13% of theory) of the highly polar (2S) -isomer were obtained as a colorless powder You. [Melting point: 239-240 ° C (decomposition); R_f= 0.13 (dichloromethane: methanol 95 : 5) ； MS (FAB): m / z = 890 (M + H)⁺. ]   1-{(2R, S, 4S, 5S) -5- (S-valinylamino) -6-cyclo Hexyl-4-hydroxy-2- (1-methyl) ethyl-hexanoyl} -S- Isoleucinyl-2-pyridylmethylamide dichloride is prepared as follows 505 mg (0.75 mmol) in 4.6 ml of 4N gaseous hydrogen chloride / anhydrous dioxane solution. ) 1-{(2R, S, 4S, 5S) -5- [N- (tert-butoxycarbonyl ) -S-Vinylamino] -6-cyclohexyl-4-hydroxy-2- (1- Methyl) ethyl-hexanoyl} -S-isoleucinyl-2-pyridylmethyla The solution of amide is stirred at 0 ° C. for 1 hour. Then 10 ml of toluene are added and the mixture is diluted. Concentrate in air. This process is repeated twice more, the residue is triturated with ether and suctioned Filter and dry under vacuum on KOH. 405 mg (84% of theory) of 1-{(2R , S, 4S, 5S) -5- (S-Vinylamino) -6-cyclohexyl-4- Hydroxy-2- (1-methyl) ethyl-hexanoyl} -S-isoleucinyl 2-Pyridylmethylamide dichloride is obtained as a colorless powder. [Melting point: 17 7-179 ℃ (decomposition); R_f= 0.38 (acetonitrile / water 9: 1); MS (FAB): m / z = 574 ( (M + H)⁺. ]   1-{(2R, S, 4S, 5S) -5- [N- (tert-butoxycarbonyl) -S-valinylamino] -6-cyclohexyl-4-hydroxy-2- (1-me Cyl) ethyl-hexanoyl} -S-isoleucinyl-2-pyridylmethylami Is prepared as follows: 239 mg (1.10 milliliters) in 10 ml anhydrous dichloromethane. Mol) N- (tert-butoxycarbonyl) -S-valine and 149 mg (1.10 millimolar) Solution of HOBT at 0 ° C. was treated with 434 mg (1.05 mmol) of DCC and the mixture was treated with 5 Stir for a minute. Then in 10 ml of dichloromethane 0.55 g (1.00 mmol) of 1-{(2R, S, 4S, 5S)-[5-amino-6 -Cyclohexyl-4-hydroxy-2- (1-methyl) ethyl-hexanoyl ] -S-Isoleucinyl-2-pyridylmethylamide dihydrochloride [EP 437 729] And 0.44 ml (4.00 mmol) of a solution of N-methylmorpholine are added dropwise. cooling The bath is removed and the reaction mixture is stirred at room temperature for 8 hours. The reaction is completed by thin layer chromatography. Determined by graphy. The resulting urea was removed by filtration and the filtrate was vacuumed. Concentrate and chromatograph on 45 g of silica gel (dichloromethane: The crude product is purified with methanol 9: 1). 507 mg (75% of theory) of 1- { (2R, S, 4S, 5S) -5- [N- (tert-butoxycarbonyl) -S-va Linylamino] -6-cyclohexyl-4-hydroxy-2- (1-methyl) e Cyl-hexanoyl} -S-isoleucinyl-2-pyridylmethylamide is colorless Obtained as a powder. [Melting point: 187 ° C (decomposition); R_f= 0.39, 0.44 (dichlorometa Methanol / methanol 9: 1); MS (FAB): m / z = 674 (M + H)⁺. ]   Inhibitor 22 is prepared as follows: 2 ml of dry dichlorometa cooled to 0 ° C. 122 mg (0.22 mmol) of EP 528 242 in ethanol (the disclosure of which is hereby incorporated by reference) Compound of Example XLII on page 62 and 26 mg (0.22 mmol) of 1- Add 28 μl (0.22 millimolar) to a stirred solution of methyl-1H-tetrazole-5-thiol. ) Of boron trifluoride etherate. Stir this mixture at 0 ° C for 45 minutes Then 10 ml ethyl acetate and 10 ml saturated NaHCO 3._ThreePoured into a mixture of aqueous solutions. Existence The phases were separated and 10 ml of saturated NaHCO 3 was added._ThreeWash with aqueous solution and water, and MgSO_FourDried on Was. The solvent was distilled off under reduced pressure and the residue was chromatographed on 50 g of silica gel. Purification with (dichloromethane: methanol 95: 5) yields 45 mg as colorless crystals. (31%) inhibition Agent 22 was obtained. [Melting point: 178 ° C (decomposition); R_f= 0.18 (dichloromethane / methano 95: 5): MS (FAB): m / z = 660 (M + H)⁺:¹H-NMR (250MHz, CD_ThreeOD) δ = 3.88 (s , 3H, NCH_Three), 5.01 (s, 2H, PhCH₂O), 7.2 (m, 10H, Ph). ]   Inhibitor 23 is prepared as follows: 100 mg (0.11 mm) in 5 ml methanol. Mol) 1-{(3RS, 4S) -4- [N- (ethoxycarbonyl) -S-phenyl Phenylalanyl-S-histidylamino] -5-cyclohexyl-3-hydroxy Cipentanoyl} -S-leucyl-3- [N- (benzyloxycarbonyl) a Minomethyl] benzylamide [prepared by standard methods for peptide synthesis] in 100 mg % Pd / C and 150 mg (2.4 mmol) ammonium formate with stirring at 60 ° C for 4 hours. You. After filtration and concentration, the residue is taken up in dichloromethane and washed twice with brine. , Na₂SO_FourDry on top. Fluffy white on removal of solvent and lyophilization 60 mg (71%) of inhibitor 23 are obtained as a powder. [R_f= 0.42, 0.45 (dichloro Methane / methanol / concentrated aqueous ammonia 9: 1: 0.1); MS (FAB): m / z = 788 (M + H)⁺ ].   Inhibitor 24 is prepared as follows: 2.60 g (2.7 milliliters) in 100 ml of methanol. Mol) of 1-{(3RS, 4S) -4- [N- (tert-butoxycarbonyl)- S-tyrosyl-S-isoleucylamino] -5-cyclohexyl-3-hydroxy Cipentanoyl} -S-leucyl-3- [N- (benzyloxycarbonyl) a Minomethyl] benzylamide [prepared by standard methods of peptide synthesis] at 300 mg Hydrogenate in the presence of% Pd / C at room temperature and atmospheric pressure for 5 hours. After filtration and concentration , Dichloromethane / methanol / concentrated aqueous ammonia (15: 1: 0.1 → 9: 1: 0.1) The residue was purified by chromatography on silica gel, eluting with 1.71 g (76%) of inhibitor 24 is obtained as a white solid. [R_f= 0.26 (dichlorometh Tan / Methanol / concentrated aqueous ammonia 9: 1: 0.1); MS (FAB): m / z = 823 (M + H)⁺. ]   Compounds 25 and 25 correspond to EP 472 078 (corresponding to USP 5,147,865) and EP 528 242, respectively. Known from. These compounds are different aspartic proteases (HIV Although it is an inhibitor of rotase), it is not Not active against Psin D (IC₅₀> 1 μM) and they are cellular amyloid Does not inhibit release. Therefore, those compounds are included as negative controls.Example 14. Inhibition of βA4 in cell culture   Cell culture: Full length transfer of APP695 cDNA (Kang et al., 1987, Nature, 325: 733-736) DNA of the pCEP4 construct (Invitrogen Corp., San Diego, CA) containing the open reading frame Transfection of HEK293 cells using the Glyco / lipofection mixture (Gibco / BRL) A cactant was made. Safe due to vector-mediated hygromycin resistance A constant cell line was selected.   Preparation of CHO cell vectorOf the APP695 cDNA from FC-4 (Kang et al., Supra) Fill the 2.36kb NruI / SpeI fragment with the large fragment of E. coli DNA polymerase I. Sm of KS Bluescript M13 + vector (Stratagene, La Jolla, CA) A blunt end was inserted into the aI cloning site to create pMTI-5. Then oligo : 5'-ctc tag aac tag tgg gtc gac acg atg ctg ccc ggt ttg-3 '(SEQ ID NO: 8 Site-directed mutagenesis (Kunkel et al., 1987, Methods in Enzymology) , 154: 367) to create a new Kozak consensus DNA sequence to generate pMTI-39. p Digest MTI-39 with NotI / HindIII and then gel-purify the 2.36 kb APP695 cDNA fragment. PcDNAINeo manufactured and cleaved with NotI / HindIII^r(Invitrogen) APP695 We generated pMTI-72 whose expression is under the control of the CMV promoter. Stable Generation of the CHO cell line is as described in Example 1, Method 1 (ii).   Preparation of vector for HEK293 cells: 2.8 kb SmaI / HindIII fragment of APP695 cDNA A piece of pSP65 / APP695 cDNA (prepared by Dyrks et al., 1988, EMBOJ., 7: 949-957) From. Digest pCEP4 vector (Invitrogen) with PvuII / HindIII, then Was ligated with the 2.8 kb APPSmaI / HindIII fragment to give pCEP / 695. This plus Expression of the APP cDNA in the mid is under the control of the CMV promoter.   Cell line maintenance: HEK293 cells have 5% CO₂， 95% humidity, DMEM ， 10% fetal bovine Serum, 50 units Pen / Strep and 2mM glutamine (BRL / Gibco) maintained at 37 ℃ .   CHO cell line is 5% CO₂, Under 95% humidity, αMEM, 10% fetal bovine serum, 50 units Pe Maintained in n / Strep and 2 mM glutamine (BRL / Gibco) at 37 ° C.   All media were purchased from Gibco / BRL and JRH Biosciences.   On the day of the experiment, cells were split into 60 mm dishes to reach a peripheral density of approximately 80%. Transfer The drug (stock solution in DMSO) was added to a final concentration of 10 μM 8 hours after planting and Incubated in a cubator for 14-16 hours. The next day, remove the medium and preheat PBS (1mM KH₂PO_Four， 10mM Na₂HPO_Four, 137 mM NaCl, 2.7 mM KCl, pH 7.4) Cells were washed twice, starved to death in methionine-free DEM medium for 30 minutes, then 15 0 μCi³⁵Labeled with S-methionine (Amersham) for 3 hours (all in the presence of drug) ). Place the cell culture dish on ice, remove the conditioned medium (4 ° C) and allow the cell debris to settle. (5 minutes at 15,000g at 4 ° C), and add the supernatant to the next protease inhibitor (1 μg / ml leupe). Putin, 0.1 μg / ml pepstatin A, 1 mM PMSF, 2 μg / ml aprotinin) Contains 1x RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate) , 0.1% SDS, 50mM Tris-HCl, pH8.0) and incubate at 100 ℃ for 5 minutes. Bate and cool again.   Immunoprecipitation1 ml of conditioned medium was mixed with 15 μl of normal mouse serum and 150 μl of Omnisorb (C albiochem) (pre-incubated with shaking at 4 ° C. for 2 hours). Slurry was removed by centrifugation (4 ° C, 10,000 g for 5 minutes), and 10 μl of monochrome Ink with null antibody 286.8A (recognizes epitopes "1-7" of βA4 sequence) It was incubated (4 ° C, 16 hours). The bound immune complex was treated with Omnisorb (150 μl) Pre-incubation together (4 ° C. for 2 hours) followed by sedimentation ((4 ° C., 10.0) 5 minutes at 00g). The immunoprecipitate was washed with ice-cold washing buffer C (10 mM Tris pH8.0, 150 mM NaCl). Was washed twice for 5 minutes each. Immune complex in 2x Tris / Tricine sample buffer Resuspend in water, boil (10 min at 100 ° C), and reduce to 16.5% or 10 ~ 20% Tris / Tricine-SDS / PAGE (Schagger and Jagow, 1987, Anal. Biochem ., 166: 368). The gel is fixed, dried and reinforced (Amplify, Amersha m), followed by Fuji Phospo-imager^TMExposed to plate and Kodax X-ray film (-70 ° C) .   result: Anti-APP monoclonal antibody 22Cll (Weidemann et al., 1989, Cell, 7: 115- (Prepared by P.126) and paired with the cytoplasmic region of APP. Immunoprecipitation using the anti-APP-C terminal antibody 91.07 elicited by the method (Example 6 (ii)). One of the HEK293 transfectants, as demonstrated by Clone (293 / 695.9) is better than that reported for CHO / 695 cells. It showed a higher APP695 expression. Both cell lines paired with bacterial APP fusion proteins. Raised polyclonal rabbit antibody 45.7 (prepared by Weidemann et al., Supra) ) Proved by immunoprecipitation As a result, most of these APP pools are rapidly diluted in extracellular medium (conditioned medium). Secreted quickly.   To detect βA4 released in the conditioned medium, the cells were³⁵With S-methionine Radiolabeled and labeled proteins released by cells in conditioned medium were Immunoprecipitation with clonal antibody 286.8A. Both cell lines are 16.5% Tris / Tric Approximately 4 kDa immune response in conditioned medium on fluorogram of ine polyacrylamide gel Shows a responsive band which is exposed on the Phosho-imager plate for about 2 hours Visible after or after an exposure time of 24 hours. In addition, monoclonal antibody 286.8A Has an apparent molecular weight of 102 kDa (determined for the CHO / 695 cell line) The protein is precipitated, which is at the C-terminus by so-called α-secretase. It represents a secreted APP (αAPP) cleaved by. The essence of the 4kDa band is , ΒA4 sequences "2-43" (rPAb 63122) and "1-40" (rPAb 3572). Two polyclonal rabbit antibodies raised and one for βA4 sequence "17-24" Monoclonal antibody (4G8; Kim et al., 1988, Neur. Res. Commn., 2: 121-130) It was confirmed by three additional independent antibodies consisting of: Furthermore, the nature of βA4 and Other sizes are translated in vitro in parallel and radiolabeled (³⁵S-methionine) It was confirmed by experimenting with the obtained βA4 "1-42" peptide. Antibody 4G8, 63122 And R3572 also precipitated a peptide with approximately the same strength as βA4 near 3 kDa. Was. This signal is transmitted by α-secretase (α-cleavage; βA4 +16/17 position). ) And at the C-terminus of βA4 (showing γ-cleavage; after positions + 39-43 of βA4 (See also Figure 25) derived from the cleaved precursor molecule. Previously, this fragment was p3 "(Haas et al., 1992, Nature, 359: 322-325). Therefore, two Transfected cells produce significant levels of βA4 It can be concluded that it produces and releases it into the medium in a relatively short time. Wear.   Inhibition of βA4 secretion using these two transfected cell lines The ability of the compound (Example 13) was evaluated. The cells were incubated with 10 μM inhibitor for 16 hours. I had a cube. The cells are then washed with serum-free medium and metabolically in the presence of inhibitor.³⁵ Labeled with S-methionine for 3 hours. Phospho-imager intensity of βA4 signal^TM It was quantified by analysis.   Six of the 24 selected cathepsin D in vitro inhibitors were identified as HEK29 Reduced βA4 levels to less than 50% in a cellular assay using 3/695 cells Was identified. Repeat this experiment at least twice to cause a significant reduction The values for the different compounds are given in Table 8. Cathepsin D quite strong in in vitro assay Structurally very similar compounds that showed no activity, such as inhibitors # 25 and # 26 , Also tested for activity in a cell assay system. These latter two compounds are , Did not significantly affect βA4 levels as shown in Table 8. High resolution Phospho-imager at about 100 kDa on gel^TMWhen evaluated by signal intensity The amount of secreted APP did not change significantly. No change in overall cell morphology Therefore, none of the tested inhibitors affected cell viability. More cells As an indicator of viability, an inhibitor that inhibited βA4 production in cells was To test their effects on the conversion of zolium salts MTT to formazan (CellTiter96^TMAssay, Promega). 67 control value with inhibitor # 10 There is no significant change in the titer of cells that reduce MTT, except for a slight decrease in%. I couldn't guess.   From the above, the six inhibitors identified in Example 6 were tested in their cellular assay. It will be clear that it shows a significant effect in the system. All six compounds reduced secreted βA4 levels by more than 50% compared to controls . A decrease in βA4 was confirmed by an additional independent anti-βA4 antibody. Furthermore, cell life Existence tests showed no measurable difference except for # 10. Therefore, 6 identifications Compound blocked the production / accumulation of βA4 subunits that form amyloid plaques. The harm will prove to be therapeutically beneficial.   The specification and claims are provided by way of illustration and not limitation. It should be apparent that various modifications and changes can be made without departing from the spirit and scope of the present invention. It will be white. In particular, the additional inhibitors disclosed in the above applications are also described herein. It will be useful as listed below. The claims also cover their alternative embodiments Intend.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI A61K 31/535 9454-4C A61K 31/535 38/55 AAM 9152-4B C12N 9/99 C12N 9/99 7823- 4B C12Q 1/37 C12Q 1/37 9284-4C C07D 211/38 // C07D 211/38 9051-4C 261/02 261/02 9159-4C 409/12 213 409/12 213 9159-4C 409/14 409 / 14 9356-4H C07K 5/03 C07K 5/03 0276-2J G01N 33/53 D G01N 33/53 9051-4C A61K 37/64 AAM (72) Inventor Benz, Gintel Hans Heinz Hellbel Connecticut, USA 06437, Guilleford , Sylvan Hills Rose 81 (72) Inventor Hebig, Dieter Germany, Day-42096 Wuppertal Kurumahha 82 (72) inventor Dreyer, Robert Norman United States, Connecticut 06492, Wallingford, East Center Street, 838 (72) inventor Koenig, Gerhard United States, Connecticut 06405, Branford Three Elm Road 10

Claims (1)

[Claims] 1. A method of regulating the formation of β-amyloid protein using an inhibitor of at least one protease specific for Alzheimer's β-amyloid protein precursor. 2. The method of claim 1, wherein the inhibitor is selected from the group of inhibitors consisting of those specific for aspartic proteases and serine proteases. 3. 3. The method of claim 2, wherein the aspartic protease inhibitor specifically inhibits cathepsin D. 4. The serine protease inhibitor specifically inhibits serine proteases that are inhibited by α-2-antiplasmin, chymotrypsin inhibitor II or TPCK and that form C-terminal APP fragments of 11, 14 and 18 kDa at pH 7-9, The method of claim 2. 5. The inhibitor is selected from 1-deoxynojirimycin, diazoacetyl-norleucine methyl ester, Gly-Glu-Gly-Phe-Leu-Gly-Asp-Phe-Leu (SEQ ID NO: 6), Ascaris pepsin inhibitor and pepstatin. The method of claim 3 selected from the group consisting of: 6. The inhibitor is The method of claim 1, selected from the group consisting of: 7. A method of preventing amyloid plaque formation in Alzheimer's disease comprising administering a therapeutic amount of a cathepsin D inhibitor. 8. Transition state analogs wherein the inhibitor contains a reactive spacer selected from the group consisting of reduced amides, hydroxy isosteres, ketone isotopes, dihydroxy isosteres, statin analogs, phosphonates, phosphonamides and reverse amides. The method of claim 7, which is the body. 9. The inhibitor is 8. The method of claim 7, selected from the group consisting of: Ten. A method of identifying an inhibitor of cathepsin D, comprising: (a) a first incubation in which cathepsin D is incubated with a peptide substrate that can be cleaved by cathepsin D, and cathepsin D is treated under conditions that are catalytically active. (B) incubating cathepsin D with a peptide substrate capable of being cleaved by cathepsin D in the presence of a potential inhibitor to produce a second incubation; (c) producing peptide formed over a period of time. The amount of product is analyzed and the rate of product formation during the first and second incubations is calculated; and (d) the reduction in enzyme activity observed in the presence of the potential inhibitor is calculated, where A method comprising a reduction exhibiting inhibitory activity. 11. 11. The method of claim 10, wherein the cathepsin D is human cathepsin D. 12． 11. The method of claim 10, wherein the peptide substrate is N-dansylated. 13. A method for measuring the proteolytic activity of a molecule capable of degrading an amyloid precursor protein, comprising: (a) obtaining a physiological sample from human; (b) catalyzing the amyloid precursor proteolytic protease in the sample. The sample is incubated in the presence of an amyloid precursor protein substrate under conditions that are biologically active; (c) making a gel using a portion of the finished incubation mixture of step (b); (d) Electrophoresing the gel of step (c) to obtain an electrophoretic pattern representing different peptide components; (e) blotting the components of step (d) onto a membrane; (f) the steps from step (e) Contacting the blot membrane with an anti-amyloid precursor protein antibody; (g) reacting the blot membrane with a secondary antibody that recognizes the anti-amyloid precursor protein antibody, wherein the secondary antibody is detectable It is coupled to a ligand; and (h) the method comprising examining the staining intensity of blots in the region corresponding to the sufficient size fragment to contain the β- amyloid peptide sequence. 14. 14. The method of claim 13, wherein the physiological sample is selected from the group consisting of brain tissue and cerebrospinal fluid. 15. 15. The method of claim 14, wherein the physiological sample contains cathepsin D. 16． 14. The method of claim 13, further comprising treating the physiological sample to obtain a crude homogenate, a soluble fraction or a detergent-solubilized membrane fraction. 17． 14. The method of claim 13, wherein the amyloid precursor protein substrate is translated from a gene sequence containing a point mutation. 18. 14. The method of claim 13, wherein the amyloid precursor protein substrate corresponds to the C-terminal portion of the amyloid precursor protein. 19. 14. The method of claim 13, wherein the anti-amyloid precursor protein antibody recognizes a peptide selected from the group consisting of a C-terminal fragment of amyloid precursor protein and β-amyloid peptide. 20. 20. The method of claim 19, wherein the C-terminal fragment comprises a C-100 or β-amyloid peptide fragment as detected by co-migration with a recombinant C-100 or β-amyloid molecular weight marker. twenty one. A method for identifying a protease inhibitor specific for an amyloid precursor protein, comprising: (a) obtaining a physiological sample from a human; (b) a portion of said sample and a selected amyloid precursor protein substrate. (C) predetermined to form a first incubation and to form a second incubation using the second portion of the sample, the selected amyloid precursor protein substrate and a test inhibitor; After a period of time, the incubation of step (b) is terminated; (d) a portion of the reaction mixture that was completed in step (c) is used to make a gel; (e) the gel of step (d) is electrophoresed; Obtaining an electrophoretic pattern representing the separate peptide components; (f) blotting the components of step (e) onto a membrane; (g) contacting the blot membrane from step (f) with an anti-amyloid precursor protein antibody (H) above The blot membrane is reacted with a secondary antibody that recognizes the anti-amyloid precursor protein antibody, wherein the secondary antibody is bound to a detectable marker; (i) sufficient to contain a β-amyloid sequence. Examining the intensity of staining of the blot in regions corresponding to different size fragments; and (j) comparing the intensity of bands of the same size observed from the first and second incubations. twenty two. 22. The method of claim 21, wherein the protein specific for the amyloid precursor protein is cathepsin D.