Patent Publication Number: US-2005142612-A1

Title: Inhibitors of amyloid precursor protein processing

Description:
BACKGROUND OF THE INVENTION  
      1. Field of the Invention  
      The present invention relates to inhibitors of amyloid precursor protein (APP) processing. The invention also relates to treating the symptoms of Alzheimer&#39;s disease by applying the inhibitors to the person in need thereof.  
      2. General Background and State of the Art  
      Alzheimer&#39;s disease (AD), the most common cause of dementia in elderly people, is a complex disorder of the central nervous system clinically characterized by a progressive loss of cognitive abilities. Pathological hallmarks of AD are extracellular senile plaques, intracellular neurofibrillary tangles composed of abnormal tau paired helical filaments, loss of neurons, cerebral amyloid angiopathy, and degeneration of cerebrovasculatures in certain areas of the brain (Marti et al.,  Proc Natl Acad Sci USA  1998; 95(26):15809-15814; Yamada M.,  Neuropathology  2000; 20(1): 8-22; Yankner B A,  Neuron  1996; 16(5):921-932). β-amyloid (Aβ) is the major component of senile plaques and is derived from the amyloid precursor protein by proteolytic cleavage (Vassar et al., Neuron 2000; 27(3): 419-422). Although accumulating evidence suggests that Aβ is a key causative agent of AD (Calhoun et al., Nature 1998; 395(6704):755-756; Hardy et al.,  Science  1992; 256(5054):184-185; Hsiao et al.,  Science  1996; 274(5284):99-102; Lewis et al.,  Science  2001; 293(5534):1487-1491; Schenk et al.,  Nature  1999; 400(6740):173-177; Sommer B.,  Curr Opin Pharmacol  2002; 2(1):87-92; Thomas et al.,  Nature  1996; 380(6570):168-171), the exact mechanism of neuronal degeneration in AD is not clear. However, it is likely that multiple factors are involved in the development of the disease.  
      Alzheimer&#39;s disease (AD) is a progressive neurodegenerative dementia afflicting 1% of the population over age 65. A significant pathological feature, however, is an overabundance of diffuse and compact senile plaques in association and limbic areas of the brain. Although these plaques contain multiple proteins, their cores are composed primarily of β-amyloid, a 40-42 amino acid proteolytic fragment derived from the amyloid precursor protein (Selkoe D J. Cellular and molecular biology of β-amyloid precursor and Alzheimer&#39;s disease. In: Prusiner S B, Rosenberg R N, Mauro S D, et al, eds. The molecular and genetic basis of neurological disease. Boston: Butterworth Heinemann Press, 1997:601-602).  
      APP is a single-transmembrane protein with a 590-680 amino acid long extracellular amino terminal domain and an approximately 55 amino acid cytoplasmic tail which contains intracellular trafficking signals. mRNA from the APP gene on chromosome 21 undergoes alternative splicing to yield eight possible isoforms, three of which (the 695, 751 and 770 amino acid isoforms) predominate in the brain. APP 695  is the shortest of the three isoforms and is produced mainly in neurons. Alternatively, APP 751 , which contains a Kunitz-protease inhibitor (KPI) domain, and APP 770 , which contains both the KPI domain and an MRC-OX2 antigen domain, are found mostly in non-neuronal glial cells. All three isoforms share the same Aβ, transmembrane and intracellular domains and are thus all potentially amyloidogenic. The normal function of APP is currently unknown, although in neurons it has been demonstrated to be localized in synapses where it may play a role in neurite extension or memory.  
      APP can undergo proteolytic processing via 2 pathways. Cleavage by α-secretase occurs within the Aβ domain and generates the large soluble N-terminal APPα and a non-amyloidogenic C-terminal fragment. Further proteolysis of this fragment by γ-secretase generates yet other the non-amyloidogenic peptide p3. Alternatively, cleavage of APP by β-secretase occurs at the beginning of the Aβ domain and generates a shorter soluble N-terminus, APPβ, as well as an amyloidogenic C-terminal fragment (C99). Further cleavage of this C-terminal fragment by γ-secretase generates Aβ. Cleavage by γ-secretase or multiple γ-secretases can result in C-terminal heterogeneity of Aβ to generate Aβ40 and Aβ42.  
      In further detail, APP is trafficked through the constitutive secretory pathway, where it undergoes post-translational processing including a variety of proteolytic cleavage events. APP can be cleaved by three enzymatic activities termed α-, β-, and γ-secretase ( FIG. 1 ). α-secretase cleaves APP at amino acid 17 of the Aβ domain, thus releasing the large amino-terminal fragment sAPPα for secretion. Since α-secretase cleaves within the Aβ domain, this cleavage precludes Aβ formation. Rather, the intracellular carboxy-terminal domain of APP generated by α-secretase cleavage is subsequently cleaved by γ-secretase within the predicted transmembrane domain to generate a 22-24 residue (3 kD) fragment termed p3 which is non-amyloidogenic (Sisodia et al.,  Science;  248:492-5 (1990)). Alternatively, APP can be cleaved by β-secretase to define the amino terminus of Aβ and to generate the soluble amino-terminal fragment APPβ. Subsequent cleavage of the intracellular carboxy-terminal domain of APP by γ-secretase yields full-length Aβ. Carboxy-terminal cleavage of Aβ by γ-secretase results in the generation of multiple peptides, the two most common being 40-amino acid Aβ (Aβ40) and 42-amino acid Aβ (Aβ42). Aβ40 comprises 90-95% of secreted Aβ and is the predominant species recovered from cerebrospinal fluid (Seubert et al.,  Nature;  359:325-7 (1992)). In contrast, less than 10% of secreted Aβ is Aβ42. Despite the relative paucity of Aβ42 production, Aβ42 is the predominant species found in plaques and is deposited initially (Iwatsubo et al., Neuron; 13:45-53 (1993)), perhaps due to its ability to form insoluble amyloid aggregates more rapidly than Aβ40 (Jarrett et al.,  Biochemistry;  32:4693-7 (1993); Jarret et al.,  Cell;  73:1055-89 (1993)).  
      Aβ has been postulated to be a causal factor in the pathogenesis of AD. The presence of Aβ-containing amyloid plaques is necessary for the neuropathological diagnosis of AD, suggesting that these entities may be involved in the etiology of the disease. Supportive evidence for the causal role of Aβ in AD can be found in patients with Down&#39;s syndrome, who often develop AD-like symptoms and pathology after age 40 (Wisniewski et al.,  Neuron;  35:957-61(1985)). Down&#39;s syndrome patients produce elevated APP presumably due to an additional copy of chromosome 21 and exhibit florid AD-like amyloid plaques prior to the onset of other AD symptoms, suggesting that amyloid deposition is an initial event (Giaccone et al.,  Neurosci Lett;  97:232-8 (1989)). Furthermore, alterations in APP processing have been linked to a subset of familial AD patients (FAD) with autosomal dominant mutations in APP (Goate et al.,  Nature;  349:704-6 (1991); Citron et al.,  Nature;  360:672-4 (1992)), presenilin 1 (PS1; 14) and presenilin 2 (PS2; 15).  
      Given the evidence that altered production of Aβ may be an initial event in the development of AD, much research has focused on understanding the mechanisms by which APP is processed to generate Aβ. The main cleavage pathways appear to be conserved in both neuronal and non-neuronal cells, but the predominant intracellular sites of production and the particular products formed are cell-type dependent. Non-neuronal cells preferentially process APP via α- and γ-secretase cleavage to generate APPα and the non-amyloidogenic fragment p3. Thus, non-neuronal cells are not a significant source of Aβ under normal conditions. However, although non-neuronal cells predominantly utilize α-secretase, neurons do not rely heavily on this pathway and produce very low levels of p3 (Chyung et al.,  J Cell Bio;  138:671-80 (1997)). Regardless of the cell type, α-secretase cleaves APP constitutively (Sisodia et al.,  Science;  248:492-5 (1990)) and is thought to occur mainly at the cell surface since APPα cannot be detected intracellularly (Chyumg et al.,  J Cell Bio;  138:671-80 (1997); Forman et al.,  J Biol Chem;  272:32247-53(1997)) and cell-surface labeled APP can be recovered as APPα in the medium (Sisodia,  Proc Natl Acad Sci USA;  89:6075-9 (1992)). Cleavage by β- and γ-secretases yields Aβ and is also a constitutive event, as Aβ can be detected in normal brains in picomolar to nanomolar concentrations (Haass et al.,  Nature;  359:322-5 (1992); Seubert et al.,  Nature;  361:260-3 (1993)).  
      It can be seen that one of the ways to prevent the accumulation of β-amyloid is to prevent β-secretase and/or γ-secretase from cleaving and processing APP. However, secretases are involved in the processing of many important proteins in the organism, and therefore inhibiting secretase activity may cause undesirable side effects. Thus, inactivating β-secretease and/or γ-secretase per se is not an appealing method of preventing APP processing.  
      Therefore, there is a need in the art to provide a method of treating or preventing Alzheimer&#39;s Disease, and in particular inhibiting β-amyloid formation and aggregation. Further, it is desirable to develop compounds that inhibit the processing of APP only without affecting other cellular machinery. Furthermore, design of APP specific inhibitors that can bind to the β-secretase and/or γ-secretase site of APP is desirable to block the approach of these secretases avoiding the processing of other important substrates of these secretases.  
     SUMMARY OF THE INVENTION  
      The invention provides solutions to the above-mentioned problems. The present invention is based on the discovery of several polypeptides that bind to the β- or γ-secretase cleavage sites on APP. Particularly exemplified are various decamers, although the invention is not limited to decamers. The invention is directed to any polypeptide or peptide mimetic compound that binds to the β- or γ-secretase cleavage sites on APP, including polypeptides or peptide mimetics having about 4 to 20 amino acids, in particular, about 4-15 amino acids, and further in particular 4 to 11 amino acids, and still in particular, 4-7 amino acids. Further, mimetics that cross the blood-brain barrier are also contemplated. Furthermore, the compounds to be used as drug should possess high affinity and specificity for APP, be stable, small and able to be transported across the plasma membrane with adequate solubility and hydrophobicity.  
      In certain respects, the present invention is directed to a polypeptide or a peptide mimetic compound which binds to the β-secretase cleavage site of amyloid precursor protein. The polypeptide or the peptide mimetic compound may contain about 4 to 20 amino acids long. The polypeptide may contain about 4 to 15 amino acids or about 4 to 10 amino acids. In another aspect, the invention is directed to a compound that binds to the P-secretase cleavage site of amyloid precursor protein and contains about 4 to 20, 4 to 15 or 4 to 10 amino acids.  
      The β-secretase cleavage site of the amyloid precursor protein may be located within SEVKMDAEFR (SEQ ID NO:1), which is the wild-type version. However, the invention contemplates and includes non-wild type β-secretase cleavage sites, such as SEVNLDAEFR (SEQ ID NO:2), which is an exemplified mutant sequence. The cleavage products of the amyloid precursor protein having the sequence of SEVKMDAEFR (SEQ ID NO:1) or SEVNLDAEFR (SEQ ID NO:2) may be SEVKM (SEQ ID NO:3) and DAEFR (SEQ ID NO:4); or SEVNL (SEQ ID NO:5) and DAEFR (SEQ ID NO:4), respectively.  
      In one aspect of the invention, the polypeptide which binds to the wild type β-secretase cleavage site of amyloid precursor protein may comprise various fragments of SEFCIHLHFR (SEQ ID NO:6) or SEFCIQIHFR (SEQ ID NO:7). However, other polypeptides and peptide mimetic compounds thereof may be synthesized against the wild-type and non-wild type β-secretase cleavage site based on known peptide complementarity and known chemical synthesis methods. Thus, in one aspect of the invention, the polypeptide may be translated from complementary nucleic acid sequence that encodes the β-secretase cleavage site. Other peptide mimetic compounds are also contemplated in the invention based on making mutations and synthesizing an array of biomimetic compounds that are intelligently based on the peptide sequence.  
      The invention is further directed to a method of preventing binding between APP and β-secretase, comprising providing a compound which inhibits the interaction between APP and β-secretase such as the polypeptide or peptide mimetic compound described above. However, the compound may be any class of compound so long as it is capable of inhibiting the binding between APP and β-secretase. In the method, the compound may be provided to a mammal suffering from a disease indicated by formation of amyloid plaques.  
      The invention may include a method of screening for a compound which inhibits APP/β-secretase binding, comprising: 
          (a) contacting the compound with a sample containing APP or a fragment of APP that contains β-secretase binding site and β-secretase;     (b) determining the level of the APP or fragment of APP/β-secretase binding under conditions in which the APP or fragment of APP and β-secretase normally specifically bind to each other;     (c) determining the level of the APP or fragment of APP/β-secretase binding in the presence of the compound; and     (d) comparing the level of the APP or fragment of APP/β-secretase binding described in parts (a) and (b), wherein if the level is lower in (c) than in (b), then the compound is an inhibitor of APP/β-secretase binding.        

      The invention may also include a method of treating Alzheimer&#39;s Disease comprising administering to a person in need thereof a therapeutically effective amount of a compound which inhibits binding between APP and β-secretase.  
      Further, the invention may also include a peptide mimetic compound, which mimics the activity of the polypeptide which specifically binds to the β-secretase cleavage site of amyloid precursor protein and which may be effective in inhibiting binding between the APP and β-secretase.  
      The present invention is also directed to a polypeptide described above that binds to β-secretase cleavage site, which is covalently linked to amino acid residues that aid in transport of the polypeptide through the cell membrane such as the blood-brain barrier. In a preferred aspect, without limitation, the amino acid residues may comprise Arginine.  
      In another aspect of the invention, the present invention is directed to a polypeptide which binds to γ-secretase cleavage site of amyloid precursor protein. The polypeptide may be about 4 to 20 amino acids long. The polypeptide may be about 4 to 15 amino acids or about 4 to 10 amino acids long. In another aspect, the invention is directed to a compound that binds to the γ-secretase cleavage site of amyloid precursor protein and contains about 4 to 20, 4 to 15 or 4 to 10 amino acids.  
      The γ-secretase cleavage site of the amyloid precursor protein may be within GVVIATVIVI (SEQ ID NO:8), which is the wild-type version. However, the invention contemplates and includes non-wild type γ-secretase cleavage sites.  
      The polypeptide which binds to the γ-secretase cleavage site of amyloid precursor protein may comprise PQQYRCHRQR (SEQ ID NO:9) or a fragment thereof. In one aspect of the invention, the polypeptide may be translated from complementary nucleic acid sequence that encodes the γ-secretase cleavage site. However, other polypeptides and peptide mimetic compounds thereof may be synthesized against the wild-type and non-wild type γ-secretase cleavage site based on known peptide complementarity and known chemical synthesis methods. Other peptide mimetic compounds are also contemplated in the invention based on making mutations and synthesizing an array of biomimetic compounds that are intelligently based on the peptide sequence.  
      The invention is further directed to a method of preventing binding between APP and γ-secretase, comprising providing a compound which inhibits the interaction between APP and γ-secretase, such as a polypeptide or peptide mimetic compound described above. However, the compound may be any class of compound so long as it is capable of inhibiting the binding between APP and γ-secretase. In the method, the compound may be provided to a mammal suffering from a disease indicated by formation of amyloid plaques. Further in the method, the compound may be a polypeptide.  
      The invention may include a method of screening for a compound which inhibits APP/γ-secretase binding, comprising: 
          (a) contacting the compound with a sample containing APP or a fragment of APP that contains γ-secretase binding site and γ-secretase;     (b) determining level of the APP or fragment of APP/γ-secretase binding under conditions in which the APP or fragment of APP and γ-secretase normally specifically bind to each other;     (c) determining level of the APP or fragment of APP/γ-secretase binding in the presence of the compound; and     (d) comparing the level of the APP or fragment of APP/γ-secretase binding described in parts (a) and (b), wherein if the level is lower in (c) than in (b), then the compound is an inhibitor of APP/γ-secretase binding.        

      The invention may also include a method of treating Alzheimer&#39;s Disease comprising administering to a person in need thereof a therapeutically effective amount of a compound which inhibits binding between APP and γ-secretase.  
      Further, the invention may also include a polypeptide or peptide mimetic compound, which mimics the activity of the polypeptide which specifically binds to γ-secretase cleavage site of amyloid precursor protein and which may be effective in inhibiting binding between the APP and γ-secretase.  
      The present invention is also directed to a polypeptide described above that binds to the γ-secretase cleavage site, which is covalently linked to amino acid residues that aid in transport of the polypeptide through the cell membrane such as the blood-brain barrier. In a preferred aspect, without limitation, the amino acid residues may comprise Arginine.  
      These and other objects of the invention will be more fully understood from the following description of the invention, the referenced drawings attached hereto and the claims appended hereto. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
      The present invention will become more fully understood from the detailed description given herein below, and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, and wherein;  
       FIG. 1  shows the APP processing scheme.  
       FIGS. 2A and 2B  show processes of obtaining complementary peptides for Swedish mutant type APP ( FIG. 2A ) and wild-type APP ( FIG. 2B ). In  FIG. 2A , mRNA sequence of the P-secretase cleavage site of APPsw is depicted as 5′-ucugaagugaaucuggaugcagaauuccga-3′ (SEQ ID NO:10), which translates to the polypeptide SEFCIQIHFR (SEQ ID NO:7) (c-Sub M); and the anti-sense mRNA sequence of the β-secretase cleavage site of APPsw is depicted as 3′-agacuucacuuagaccuacgucuuaaggcu-5′ (SEQ ID NO:11), which translates to the polypeptide RLHLDLRLKA (SEQ ID NO:12) (Sub M-c). In  FIG. 2B , mRNA sequence of the β-secretase cleavage site of APP is depicted as 5′-ucugaagugaagauggaugcagaauuccga-3′ (SEQ ID NO:13), which translates to the polypeptide SEFCIHLHFR (SEQ ID NO:6) (c-Sub W); and the anti-sense mRNA sequence of the α-secretase cleavage site of APP is depicted as 3′-agacuucacuucuaccuacgucuuaaggcu-5′ (SEQ ID NO:43), which translates to the polypeptide RLHFYLRLKA (SEQ ID NO:14) (Sub W-c).  
       FIGS. 3A and 3B  show binding of substrate M ( FIG. 3A ) and substrate W ( FIG. 3B ) to their complementary peptides. In  FIG. 3A , different amounts of complementary peptides were immobilized on plastic well and biotin-labeled Substrate M was added to the well. The bound Substrate M was determined by reaction with Steptavidin-horseradish peroxidase. Control peptide refers to a decapeptide which has an unrelated sequence. In  FIG. 3B , complementary peptides were immobilized on plastic well and biotin-labeled Substrate M was added to the well. The bound Substrate M was determined by reaction with Steptavidin-horseradish peroxidase.  
       FIG. 4  shows inhibition of cleavage of Substrate M by β-secretase by complementary peptides. c-SubM CΔ1 refers to deletion of one amino acid from the C-terminus of c-SubM.  
       FIG. 5  shows deletion mutants of APPsw inhibitor used in the experiment. c-SubM (SEFCIQIHFR) (SEQ ID NO:7), c-SubM ΔN1 (EFCIQIHFR) (SEQ ID NO:15), c-SubM ΔN2 (FCIQIHFR) (SEQ ID NO:16), c-SubM ΔN3 (CIQIHFR) (SEQ ID NO:17), c-SubM ΔN4 (IQIHFR) (SEQ ID NO:18), c-SubM ΔN5 (QIHFR) (SEQ ID NO:19), c-SubM ΔC1 (SEFCIQIHF) (SEQ ID NO:20), c-SubM AC2 (SEFCIQ1H) (SEQ ID NO:21), c-SubM ΔC3 (SEFCIQI) (SEQ ID NO:22), c-SubM ΔC4 (SEFCIQ) (SEQ ID NO:23), c-SubM AC5 (SEFCI) (SEQ ID NO:24), c-SubM AC6 (SEFC) (SEQ ID NO:25), c-SubM ΔC7 (SEF).  
       FIG. 6  shows the effects of various deletion peptides on substrate cleavage.  
       FIG. 7  shows the inhibitory activities of the additional peptides with terminal deletions on β-secretase cleavage.  
       FIG. 8  shows concentration dependent inhibitory activities of various peptides tested. c-SubM ΔN2C1 (FCIQIHF) (SEQ ID NO:26), c-SubM ΔN1C1 (EFCIQIHF) (SEQ ID NO:27), c-SubM ΔC3 (SEFCIQI) (SEQ ID NO:22), c-SubM AC5 (SEFCI) (SEQ ID NO:24), c-SubW (SEFCIHLHFR) (SEQ ID NO:6), c-SubM ΔN3C3 (CIQI) (SEQ ID NO:28), c-SubM AC1 (SEFCIQIHF) (SEQ ID NO:20), c-SubM ΔN3C1 (CIQIHF) (SEQ ID NO:29), c-SubM (SEFCIQIHFR) (SEQ ID NO:7).  
       FIGS. 9A and 9B  show binding between the complementary peptides and SubM and binding between the complementary peptides and SubW, respectively.  
       FIG. 10  describes cell based assay system to be used for determination of inhibitory activities of the complementary peptides.  
       FIG. 11  shows the effects of the APP inhibitor peptides on HEK293-APP cells. Whole cell extracts were loaded. 16E10 antibody detects N-terminal of Aβ. Lanes 1.Control cells; 2.c-Sub M; 3.c-Sub M ΔC6; 4.c-Sub M ΔN1C1; 5.Control cells; 6.β-secretase inhibitor (commercial, peptide based).  
       FIG. 12  shows the effects of the APP inhibitor peptides on HEK293-APPsw cells. Whole cell extracts were loaded. 16E10 antibody detects N-terminal of Aβ. Lanes 1.Blank; 2.c-Sub M ΔC1; 3.c-Sub M ΔC5; 4.c-Sub M ΔN2C1; 5.c-Sub M ΔN3C 3; 6. β-secretase inhibitor.    
       FIG. 13  shows the activities of APP inhibitor-R 9  on rhBACE1 and fluo-Sub M system.  
       FIG. 14  shows the result of APP inhibitor-R 9  transport assay indicating the transportation of the oligoarginine-coupled APP inhibitors into the cells.  
       FIG. 15  shows the activities of APP inhibitor-R 9  on 293-APP cells.  
       FIG. 16  shows a schematic diagram of specificity assay for APPsw inhibitors.  
       FIG. 17  shows cleavage rate of β-secretase substrates at various β-secretase concentrations.  
       FIG. 18  shows inhibitory activities of APPsw inhibitor on each β-secretase substrate.  
       FIG. 19  shows process of obtaining complementary peptides for APP γ-secretase cleavage site. A γ-secretase cleavage site is depicted as GVVIATVIVI (SEQ ID NO:8). The mRNA sequence of the γ-secretase cleavage site is depicted as 5′-ggu guu guc aua gcg aca gug auc guc auc-3′ (SEQ ID NO:30), which translates to the polypeptide DDDHCRYDNT (SEQ ID NO:31) (γCh1); and the anti-sense mRNA sequence of the γ-secretase cleavage site is depicted as 3′-cca caa cag uau cgc ugu cac uag cag uag-5′ (SEQ ID NO:32), which translates to the polypeptide PQQYRCHRQR (SEQ ID NO:9) (γCh2).  
       FIGS. 20A and 20B  show γ-secretase cleavage activities in several cell lines.  FIG. 20A  shows activity in HT22 (5.55 mg/ml)—immortalized mouse hippocampal neuron and PC12 (7.61 mg/ml)—rat adrenal pheochromocytoma.  FIG. 20B  shows activity in HN33 (5.95 mg/ml)—mouse hippocampal neuron+neuroblastoma and N2a (3.65 mg/ml)−mouse neuroblastoma.  
       FIGS. 21A and 21B  show γ-secretase activities in the presence of complementary peptides. Substrate: 12.5 μM; Complementary peptide: 200 μM; γCh1 (5′→3′) DDDHCRYDNT (SEQ ID NO:31); γCh1 ΔN1: DDHCRYDNT (SEQ ID NO:33); γCh2 (3′→5′): PQQYRCHRQR (SEQ ID NO:9); γCh2-2: PQQYHCHYQ (SEQ ID NO:34). Preincubation period was 1 hr. And γ-secretase (membrane fraction) used was 3 mg/ml.  
       FIG. 22  shows inhibitory effects of the complementary peptides in the cells indicating that the tested peptides are unable to enter the cells across the membrane.  
       FIGS. 23A-23B  show Alanine scanning data for c-SubM ΔC1N3.  FIG. 23A  shows the various alanine mutants.  FIG. 23B  shows BACE inhibitory activity. C-SubM ΔC1N3 (CIQIHF) (SEQ ID NO:29), C-SubM ΔC1N3.A1 (AIQIHF) (SEQ ID NO:35), C-SubM ΔC1N3.A2 (CAQIHF) (SEQ ID NO:36), C-SubM ΔC1N3.A3 (CIAIHF) (SEQ ID NO:37), C-SubM ΔC1N3.A4 (CIQAHF) (SEQ ID NO:38), C-SubM ΔC1N3.A5 (CIQIAF) (SEQ ID NO:39), C-SubM ΔC1N3.A6 (CIQIHA) (SEQ ID NO:40). 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
      In the present application, “a” and “an” are used to refer to both single and a plurality of objects.  
      As used herein, “about” or “substantially” generally provides a leeway from being limited to an exact number. For example, as used in the context of the length of a polypeptide sequence, “about” or “substantially” indicates that the polypeptide is not to be limited to the recited number of amino acids. A few amino acids add to or subtracted from the N-terminus or C-terminus may be included so long as the functional activity such as its binding activity is present.  
      As used herein, “amino acid” and “amino acids” refer generally to all naturally occurring L-α-amino acids. However, since peptide mimetic compounds are within the purview of the invention, non-naturally occurring amino acid residues are included in the invention.  
      As used herein, in general, the term “amino acid sequence variant” refers to molecules with some differences in their amino acid sequences as compared to a reference (e.g. native sequence) polypeptide. The amino acid alterations may be substitutions, insertions, deletions or any desired combinations of such changes in a native amino acid sequence.  
      Substitutional variants are those that have at least one amino acid residue in a native sequence removed and a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.  
      Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Also included within the scope of the invention are proteins or fragments or derivatives thereof which exhibit the same or similar biological activity and derivatives which are differentially modified during or after translation, e.g., by glycosylation, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, and so on.  
      Insertional variants are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native amino acid sequence. Immediately adjacent to an amino acid means connected to either the α-carboxy or α-amino functional group of the amino acid.  
      Deletional variants are those with one or more amino acids in the native amino acid sequence removed. Ordinarily, deletional variants will have one or two amino acids deleted in a particular region of the molecule.  
      As used herein, “APP binding polypeptide” or “ABP” refers to a polypeptide that specifically binds to APP at the β- or γ-secretase cleavage site on APP. Applicants for the first time discovered various polypeptides that bind to the β- or γ-secretase cleavage site on APP, and thus it would be within the purview of a person of skill in the art to make certain variations to the sequence, which retains the capability of binding to APP. ABP excludes β- and or γ-secretase enzymes per se that retain the cleavage activity.  
      As used herein, “carriers” include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the pharmaceutically acceptable carrier is an aqueous pH buffered solution. Examples of pharmaceutically acceptable carriers include without limitation buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.  
      As used herein, “complementary” has a meaning based upon its context of usage. For example, complementary bases or nucleotides are those characteristically forming hydrogen bonds (G-C and A-T or A-U), complementary codons nucleic acids or strands thereof are hydrogen bonded polynucleotide components of a double nucleic acid strand such of that in the classically defined double helix for example complementary amino acids usually having hydropathic complementary are those directed by members of a pair of complementary codons.  
      Complementary peptides or polypeptides and their related original peptide or protein are a pair of peptides directed by complementary nucleotide or amino acid sequences, and characteristically have a binding affinity between members of a pair. Polypeptides complementary to a peptide or at least a portion of a protein, for example, have a binding affinity for the peptide or protein portion. While peptide binding affinities are incompletely understood, they may, in part at least, be explained by the concept of amphiphilic secondary structure described by Kaiser et al. ( Science;  223:249-255 (1984)).  
      The complementary polypeptide and any peptide mimetic compound thereof whose amino acid sequence is thus determined may be obtained by diverse means such as, for example, chemical synthesis, derivation from a protein or larger polypeptide containing the amino acid sequence, or, where appropriate especially for production of a naturally occurring amino acid chain, recombinant production by transforming a unicellular organism with a DNA vector to produce a transformant unicellular organism biosynthesizing the complementary polypeptide.  
      As used herein, “effective amount” is an amount sufficient to effect beneficial or desired clinical or biochemical results. An effective amount can be administered one or more times. For purposes of this invention, an effective amount of an inhibitor compound is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state. In a preferred embodiment of the invention, the “effective amount” is defined as an amount of compound capable of preventing binding of β- or γ-secretase to APP.  
      As used herein, “hydropathic complementarity”, referring to the hydropathic scores (a relative measure of hydrophilicity and hydrophobicity) of amino acids is indicated in terms of low and high hydropathy corresponding to a high hydropathy. In referring to structures comprising amino acids, they are generally referred to as peptides, polypeptides or proteins, this order designating an increase in size between, for example, dipeptides, oligopeptides, and proteins containing many hundreds of amino acids.  
      As used herein, “inhibitor” refers to a molecule that inhibits the binding of β- or γ-secretase to APP.  
      As used herein, “ligand” refers to any molecule or agent, or compound that specifically binds covalently or transiently to a molecule such as a polypeptide.  
      As used herein, “mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, and so on. Preferably, the mammal is human.  
      As used herein, “purified” or “isolated” molecule refers to biological or synthetic molecules that are removed from their natural environment and are isolated or separated and are free from other components with which they are naturally associated.  
      As used herein, the term “specifically binds” refers to a non-random binding reaction between two molecules, for example between a polypeptide or a peptide mimetic compound that binds to the β- or γ-secretase cleavage site on APP.  
      As used herein, “subject” is a vertebrate, preferably a mammal, more preferably a human.  
      As used herein, “treatment” is an approach for obtaining beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. “Treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. “Palliating” a disease means that the extent and/or undesirable clinical manifestations of a disease state are lessened and/or the time course of the progression is slowed or lengthened, as compared to a situation without treatment.  
      Screening for Compounds That Bind to APP β- or β-Secretase Cleavage Site  
      In one embodiment, the invention is directed to screening for a compound such as a polypeptide, a peptide mimetic, or chemical compound that inhibits binding of APP to β- or γ-secretase. It is expected that the inhibitor compound will treat persons suffering from diseases that are at least in part caused by the deposit of β-amyloid.  
      A fragment of APP which contains the β- or γ-secretase cleavage site may be used as a target to screen for compounds that may prevent the cleavage of this site by β- or γ-secretase. Various libraries may be used including phage display library or chemical library to screen for compounds that bind to APP and inhibit cleavage by β- or γ-secretase.  
      Inhibitor of APP/β- or γ-Secretase Binding  
      In one aspect, the invention is directed to any inhibitor molecule that is capable of interacting with APP to block the binding of β- or γ-secretase to APP. In particular, the molecule should interact with the β- or γ-secretase binding domain of APP. It is understood that the inhibitor compound may impair the interaction between the APP and β- or γ-secretase by any number of biochemical or enzymatic inhibition kinetics, such as competitive, non-competitive, or uncompetitive inhibition, so long as the compound impairs the binding of APP with β- or γ-secretase and prevents cleavage at the β- or γ-secretase cleavage site. Exemplified polypeptides that bind to a 10 amino acid fragment of APP that contains the β-secretase cleavage site include without limitation, SEFCIHLHFR (SEQ ID NO:6) and SEFCIQIHFR (SEQ ID NO:7). Exemplified polypeptides that bind to a 10 amino acid fragment of APP that contains the γ-secretase cleavage site include without limitation, PQQYRCHRQR (SEQ ID NO:9).  
      Variant and Mutant Polypeptides  
      To improve or alter the characteristics of the inhibitor polypeptide, amino acid engineering may be employed. Recombinant DNA technology known to those skilled in the art can be used to create novel mutant polypeptides including single or multiple amino acid substitutions, deletions, additions, or fusion proteins. Similar mutant polypeptides can also be produced by chemical synthesis, especially for short peptides. Such modified polypeptides can show, e.g., increased/decreased activity or increased/decreased stability. In addition, they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions.  
      Therapeutic Composition  
      In one embodiment, the present invention relates to treatment for various diseases that are characterized by the formation of β-amyloid aggregates or amyloid plaque. In this way, the inventive therapeutic compound may be administered to human patients who are either suffering from, or prone to suffer from the disease by providing compounds that inhibit the cleavage of APP to β-amyloid by binding to the β- or γ-secretase cleavage site. In particular, the disease is associated with dementia, chronic neurodegenerative disorder of the brain, loss of nerve cell, particularly in the hippocampus and cerebral cortex, reduced neurotransmitters, cerebrovascular degeneration, and/or loss of cognitive ability. Further in particular, the present invention is directed to a treatment for Alzheimer&#39;s disease. Perferably, the compound crosses the blood-brain barrier.  
      The formulation of therapeutic compounds is generally known in the art and reference can conveniently be made to Remington&#39;s Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., USA. For example, from about 0.05 μg to about 20 mg per kilogram of body weight per day may be administered. Dosage regime may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. The active compound may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intramuscular, subcutaneous, intra nasal, intradermal or suppository routes or implanting (eg using slow release molecules by the intraperitoneal route or by using cells e.g. monocytes or dendrite cells sensitised in vitro and adoptively transferred to the recipient). Depending on the route of administration, the peptide may be required to be coated in a material to protect it from the action of enzymes, acids and other natural conditions which may inactivate the ingredients.  
      For example, the low lipophilicity of the peptides will allow them to be destroyed in the gastrointestinal tract by enzymes capable of cleaving peptide bonds and in the stomach by acid hydrolysis. In order to administer peptides by other than parenteral administration, they will be coated by, or administered with, a material to prevent its inactivation. For example, peptides may be administered in an adjuvant, co-administered with enzyme inhibitors or in liposomes. Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes.  
      The active compounds may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.  
      The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, chlorobutanol, phenol, sorbic acid, theomersal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the composition of agents delaying absorption, for example, aluminium monostearate and gelatin.  
      Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterile active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.  
      When the peptides are suitably protected as described above, the active compound may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 μg and 2000 mg of active compound.  
      The tablets, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations.  
      As used herein “pharmaceutically acceptable carrier and/or diluent” includes any and all solvents, dispersion media, coatings antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.  
      It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired.  
      The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 μg to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 μg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the ingredients.  
      Delivery Systems  
      Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, receptor-mediated endocytosis. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.  
      In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including a peptide or peptide mimetic compound of the invention, care must be taken to use materials to which the protein does not absorb. In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome. In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose.  
      A composition is said to be “pharmacologically or physiologically acceptable” if its administration can be tolerated by a recipient animal and is otherwise suitable for administration to that animal. Such an agent is said to be administered in a “therapeutically effective amount” if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.  
      Mimetics  
      The use of peptides as drugs has some very attractive advantages. They can be made to be highly specific; their potency can usually be increased by simple amino acid substitution; and many exhibit very low toxicity. However, the present invention is also directed to peptide mimetics. In particular, the mimetic is directed to peptide mimetics that cross the blood-brain barrier. APP is cleaved by secretases inside the cells, most likely in trans-Golgi network and endosomal system (Huse et al.,  J. Biol. Chem.  275:33729-37 (2000); Walter et al.,  J. Biol. Chem.  276:14634-41 (2001)). Therefore, an inhibitor compound that is modified so that the compound is able to cross the cell membrane barrier, as well as the blood-brain barrier is encompassed by the present invention.  
      A peptide mimetic is defined as a non-peptide ligand that is recognized by a peptide recognition site. Such mimetics may be structurally different from the peptides. A well-known example of a peptide mimetic is morphine. This natural opioid alkaloid is a mimetic of β-endorphin, a peptide present in the human body. While this definition of a peptide mimetic characterizes a mimetic as a non-peptide ligand, many structures exist that are somewhere in between a true peptide, which is composed of natural amino acids, and a peptide mimetic. Most compounds within the spectrum of the definition are considered peptide mimetics as well. For example, a tripeptide composed exclusively of non-natural elements can be considered a peptide mimetic. Several HIV protease inhibitors are considered peptide mimetics, although they possess amide bonds and amino acids. The debate on what constitutes a peptide mimetic is still on-going, however a person of skill in the art is able to distinguish between a mimetic and a peptide. Peptide mimetics can generally be considered as improved versions of peptides. Chemical modifications on a peptide, such as the reduction of a peptide bond, can increase its enzymatic stability. Incorporating unnatural amino acids can also enhance both activity and selectivity of the peptide. The more a peptide is altered structurally and/or chemically, the more it becomes a true peptide mimetic.  
      Peptide mimetics including peptides, proteins, and derivatives thereof, such as peptides containing non-peptide organic moieties, synthetic peptides which may or may not contain amino acids and/or peptide bonds, but retain the structural and functional features of a peptide ligand, and peptoids and oligopeptoids which are molecules comprising N-substituted glycine, such as those described by Simon et al.,  Proc. Natl. Acad. Sci. USA  89:9367 (1992); and antibodies, including anti-idiotype antibodies.  
      In another aspect of the invention, the inventive compound of the invention may be made by synthetically introducing a variety of optional compounds, such as scaffolds, turn mimetics, and peptide-bound replacements. Syntheses of amino acids to the use of a variety of linear and heterocyclic scaffolds in place of the peptide backbone may be used. Chemical procedures and methods include the transient protection of charged peptides as neutral prodrugs for improved blood-brain penetration and the replacement of peptide bonds with groups such as heterocyclic rings, olefins and fluoroolefins, and ketomethylenes.  
      Hydropathic Complementarity of Amino Acid Sequence  
      According to the principle hydropathic complementarity of amino acids, the amino acid deduced by an antisense code (either 5′→3′ or 3′→5′ direction) is generally antipathic, that is, a hydrophobic amino acid sequence can be deduced from a code for a hydrophilic amino acid sequence, vice versa (Blalock and Smith,  Biochem. Biophys. Res. Commun.  121:203-207 (1984); U.S. Pat. No. 4,863,857 (1989); U.S. Pat. No. 5,077,195 (1991), the contents of which are incorporated by reference herein in their entirety in particular with regard to explaining and providing evidence for hydropathic complementarity.). The peptides, which are designed by the hydropathic complementary approach, show inverse hydropathic relationship to the peptides encoded by sense mRNA, and the designed peptide binds target protein with specificity and high affinity (Bost et al.,  Proc. Natl. Acad. Sci. USA  82:1372-1375 (1985)). There are several examples that demonstrate successful application of this approach. Antagonists of various proteins such as ACTH, ribonuclease S peptide, c-Raf protein, fibronectin, insulin, and α-chain of fibrinogen were developed based on this approach (Bost et al.  Proc. Natl. Acad. Sci. USA  82:1372-1375 (1985); Shai et al.  Biochemistry  26:669-675 (1987); Fassina et al.  J. Biol. Chem.  264: 11252-11257 (1989): Brentani et al.  Proc. Natl. Acad. Sci. USA  85:364-367 (1988); Knutson  J. Biol. Chem.  263:14146-14151 (1988): Pasqualini et al.  J. Biol. Chem.  264:14566-14570 (1989), incorporated by reference herein in their entirety.).  
      The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. The following examples are offered by way of illustration of the present invention, and not by way of limitation.  
     EXAMPLES  
     Example 1  
     Peptides that Bind to the β-Secretase Cleavage Site of APP  
      Decamer peptide sequences that contain the cleavage site of APP by β-secretase was used. The sequence is as follow: SEVKMDAEFR (SEQ ID NO:1). This wild type peptide sequence is called Substrate W. β-secretase cleaves the peptide bond between M and D and releases the following cleavage products: SEVKM (SEQ ID NO:3) and DAEFR (SEQ ID NO:4). The Swedish mutant of APP (APPsw) is cleaved by β-secretase at much higher rate than normal APP. The decamer sequence containing the cleavage site of APPsw by β-secretase is as follows: SEVNLDAEFR (SEQ ID NO:2) ( FIG. 2 ). This mutant peptide is labeled Substrate M.  
      It was previously reported that in some cases, the peptides (complementary peptide) derived from anti-sense mRNA of a target peptide can bind to the target peptide (Blalock, J. E. and Smith, E. M.  Biochem. Biophys. Res. Commun.  121, 203-207 (1984); Gho, Y. S. and Chae, C.-B.  J. Biol. Chem.  272, 24294-24299 (1997), which are incorporated by reference in their entirety). Based on this report, we designed four peptides. The anti-sense sequences were deduced from the mRNA sequences corresponding to the two decamer substrate peptides. Genetic codes were derived from the antisense RNA by reading the sequences either in 5′→3′ or 3′→5′ directions. The following decamer peptide sequences were obtained: SEFCIHLHFR (c-Sub W, SEQ ID NO:6) and RLHFYLRLKA (Sub W-c, SEQ ID NO:14) from substrate W. From Substrate M, the following two peptides were derived: SEFCIQIHFR (c-Sub M, SEQ ID NO:7) and RLHLDLRLKA (Sub M-c, SEQ ID NO:12) ( FIG. 2 ). These peptides is collectively called complementary peptides.  
      The two complementary peptides, c-Sub W and c-Sub M bind to Substrate W and M, respectively ( FIG. 3 ) and both inhibit cleavage of the Substrate M by β-secretase ( FIG. 4 ). Sub W-c and Sub M-c do not bind to the substrate ( FIG. 3 ) and do not inhibit cleavage of Substrate M ( FIG. 4 ). In our experiment, we used Substrate M mostly due to its easy cleavage by β-secretase.  
     Example 2  
     Binding of Substrates to the Complementary Peptides  
      Complementary peptides (0.2, 2, 20, and 200 μM) were dissolved in phosphate buffered saline (PBS) (pH 7.4) and fixed to microtiter wells for 5 hr at 37° C. The wells were blocked with blocking buffer (3% BSA/PBS) for 1 hr at 37° C. Either N-terminally biotinylated Substrate W or M (20 μM) in blocking buffer was added and incubated for overnight at 4° C. Streptavidin-horseradish peroxidase in blocking buffer was added to detect resulting bound substrates. The plate was incubated for 2 hr at room temperature (RT), followed by addition of 3, 3′, 5,5′-tetramethyl-benzidine (TMB) as substrate for horseradish peroxidase for color reaction.  
      In particular, for Substrate W, complementary peptides (200 μM) dissolved in phosphate buffered saline (PBS) (pH 7.4) were chemically coupled to Reacti-Bind Maleic Anhydride Activated Polystyrene wells (Pierce Biotechnology, Inc.) for overnight at room temperature (RT). Remaining active sites of the plate were inactivated by adding ethanolamine (1 M) for 1 hr at RT. The wells were blocked with blocking buffer (3% BSA/PBS) for 1 hr at RT. N-terminally biotinylated Substrate W (20 μM) in blocking buffer was added and incubated for 3 hr at RT. Streptavidin-borseradish peroxidase in blocking buffer was added to detect resulting bound substrates. The plate was incubated for 2 hr at room temperature (RT), followed by addition of 3,3′,5,5′-tetramethyl-benzidine (TMB) as substrate for horseradish peroxidase for color reaction.  
     Example 3  
     Fluorometric Assay for the Cleavage of Substrates by β-secretase  
      This assay system utilizes fluorescence resonance energy transfer (FRET) technology. Substrate M was synthesized with two fluorophores, a fluorescent donor and a proprietary quenching acceptor (purchased from a commercial source, R&amp;D Systems). The donor fluorescence energy is significantly quenched by the acceptor. Upon cleavage of substrate by β-secretase, the fluorophore is separated from the quenching group, restoring the full fluorescence yield of donor.  
      Substrate labeled with fluorophores will be called F-Substrate M (R&amp;D Systems). Recombinant human β-secretase will be called rhBACE (recombinant human β-site APP cleavage enzyme) (purchased from R&amp;D systems).  
      F-Substrate M (20 μM) was preincubated with varying concentrations of complementary peptides in assay buffer (0.1 M NaOAc, pH 4.0) for 1 hr at RT. rhBACE (70 nM) in assay buffer was added. Cleavage by rhBACE was detected by reading emitted fluorescence level.  
     Example 4  
     HPLC Analysis of the Cleavage of Substrates by β-secretase  
      Substrate M (100 μM) was preincubated with complementary peptides (2.6 mM) in assay buffer (100 μl) overnight at RT. rhBACE (140 nM) in assay buffer was added and incubated for 11 hr at RT. Cleavage products of Substrate M by rhBACE were quantitated after separation by C-18 reversed-phase column chromatography (GRACE VyDAC).  
     Example 5  
     Effect of Deletions on Inhibitory Activity of the Complementary Peptides  
      So far we have focused on c-Sub M peptide. Serial deletions were made from N-terminus or C-terminus of c-Sub M ( FIG. 5 ), and we investigated effect of the deletions on the cleavage of Substrate M. The optimum peptide/substrate ratio for inhibition on the cleavage of substrate was determined by observing the inhibition percentage at various peptide/substrate ratios ( FIG. 4 ). Subsequently, the inhibitory activity of the deletion peptides were tested at 10 inhibitor/substrate ratio and the concentration of the Substrate M was 50 μM. Deletion of the first two amino acids from the N-terminus had little effect on the activity and deletion of five amino acids from the C-terminus of c-Sub M had little effect on the inhibitory activity ( FIG. 6 ). Further deleted peptides were tested for inhibitory activity on the cleavage of Substrate M ( FIG. 7 ). Five of ten tested peptides showed considerable inhibitory activity. C-Sub MΔN3C1 (hexa peptide) and C-Sub MAN3C3 (tetra peptide) had considerable inhibitory activity.  
     Example 6  
     Concentration Dependent Inhibitory Activity of the Complementary Peptides  
      The above-mentioned peptides that have inhibitory activity were tested at various concentrations for their inhibitory activities on their mutant substrate Substrate M ( FIG. 8 ). In general, based on the inhibitory activity, the peptides may be divided into three major groups: (1) the most active group including FCIQIHF (SEQ ID NO:26), EFCIQIHF (SEQ ID NO:27) and SEFCIQI (SEQ ID NO:22); (2) the group with medium activity including SEFCI (SEQ ID NO:24), SEFCIHLHFR (SEQ ID NO:6), which shows anomalous curve possibly due to aggregation and which is a complementary peptide for the wild type substrate, and CIQI (SEQ ID NO:28), which shows anomalous curve possibly due to aggregation; and (3) the group with less activity including CIQIHF (SEQ ID NO:29) and SEFCIQIHFR (SEQ ID NO:7). The results indicate that the inhibitory activities of the peptides correlate with their concentrations showing increased inhibitory activities as the concentrations of the peptides increase.  
     Example 7  
     Binding of Complementary Peptides to Both Wild Type (Sub W) and Mutant Type Substrates (Sub M)  
      To test their binding capability, the complementary peptides were immobilized on a plate and biotin labeled substrate was applied and then after washing, the presence and amount of the bound substrate was determined.  FIG. 9A  shows that the complementary peptide c-Sub M binds its substrate Sub M.  FIG. 9B  shows that the complementary peptide c-Sub M also binds the wild type substrate Sub W efficiently. Therefore, the complementary peptide for mutant substrate binds to both the wild type and the mutant substrates.  
     Example 8  
     Cell Based Assay System  
      In the neuronal cells of the brain, APP is processed by α-secretase or β-secretase. To investigate the effects of the inhibitors on the cell, a cell based assay system was developed as described in  FIG. 10 . C-terminal fragment of APP remaining on the cell membrane was detected by Western blot. The resulting C-terminal fragments, αCTF or βCTF are further processed by γ-secretase. However, if the cells are treated with the γ-secretase inhibitor, this processing is blocked. As a result, αCTF or βCTF accumulate in the cell. If β-secretase inhibitor or APP inhibitor is added, this processing is blocked and PCTF disappears.  
     Example 9  
     APP Inhibitor Activity on HEK293-APP Cells  
      To test APP inhibitor activities in cells, three complementary peptides, c-Sub M, c-Sub MΔC6, and c-Sub MΔN1C1, which show high absorbance in binding test, were added to whole cell extracts of HEK293-APP cells (Lanes 2, 3, and 4 in  FIG. 11 ). In addition, γ-secretase inhibitor and cholesterol were added to the cells to increase PCTF level. Commercially available peptide-based β-secretase inhibitor was included as control (Lane 6,  FIG. 11 ). To detect the N-terminal fragment of β-amyloid which is a product of APP processing, 6E10 antibody was employed. As shown in  FIG. 11 , none of the inhibitors tested showed inhibitory activity on APP processing including the commercially available peptide-based β-secretase inhibitor. Recently, it has been reported that the commercially available peptide-based inhibitor had to be linked to an oligoarginine transporter peptide to have inhibitory activity against cells. Therefore, mimetic approach is adopted to produce cell permeable analogs. (Chang et al. J. Neurochemistry 2004; 89:1409-1416).  
     Example 10  
     APP Inhibitor Activity on HEK293-APPsw Cells  
      Similarly to the results shown in Example 9, the peptide inhibitors tested on HEK293-APPsw cells showed no inhibitory activity on APP processing as the levels of βCTF detected by 6E10 antibody stayed the same in the presence of the peptide inhibitors ( FIG. 12 ). These results shown in  FIGS. 11-12  suggest that these APP inhibitors have no activity on cells because they cannot pass through the cell membrane.  
     Example 11  
     APP Inhibitor-R 9  Activity on rhBACE1 and Fluo-Sub M System  
      To overcome the problem of APP inhibitor&#39;s inability to penetrate across the cell membrane, APP inhibitors were coupled with oligo-arginine (R 9  means 9 Arginines), which is known to be a transporter peptide. These coupled peptides were labeled with FITC (Fluorescein isothiocyanate) using a linker AHX (aminohexanoic acid) to investigate whether the inhibitors pass through the cell membrane. FITC-AHX-c-Sub M-R 9  and FITC-AHX-c-Sub MΔN1C1-R 9  were made.  
      To test the inhibitory activities of these oligoarginine-coupled APP inhibitors, FRET (fluorescence resonance energy transfer) enzyme assay system was used. As shown in  FIG. 13 , c-Sub MΔN1C1-R 9  showed less inhibitory activity compared to its counterpart inhibitor c-Sub MΔN1C1 lacking R 9 . C-Sub M-R 9 , especially showed no inhibitory activity. These results indicate that coupling of oligoarginine to APP inhibitors significantly decreases inhibitory activity.  
     Example 12  
     APP Inhibitor-R 9  Transport Assay and APP Inhibitor-R 9  Activity on 293-APP Cells  
      Oligo-arginine coupled APP inhibitors that were labeled with FITC were added to HEK293-APP cells to see whether the peptides pass through the cell membrane. As shown in  FIG. 14 , the oligoarginine-coupled APP inhibitors were transported into the cells.  
      After confirming the ability of the oligo-arginine coupled APP inhibitors to enter the cells, the inhibitors were applied to HEK293-APP cells overexpressing APP to test their inhibitory activities on APP processing.  FIG. 15  shows that c-Sub M-R 9  has some inhibitory activity at low concentration, but no inhibitory activity was observed at 10 μM (upper panel). c-Sub MΔN 1 C1-R 9  showed inhibitory activity in a concentration dependent manner. This inhibitor started to show significant inhibitory activity beginning from 0.1 μM (lower panel). These results indicate that oligo-arginine coupled complementary peptides may be used as APP-specific inhibitors in the APP cells.  
     Example 13  
     Specificity of Complementary Peptides APP Inhibitor  
      One of the advantages of the APP inhibitor described in the present invention is that it is a peptide or a mimetic that bind to the β-secretase cleavage site of APP, thus not affecting other β-secretase substrates. To confirm the specificity of the inventive APP inhibitor, two different types of substrates were used, APP Sub M and ST6Gal1. Both substrates are cleaved by β-secretase under normal conditions and the effect of the inventive APP inhibitor on the substrate cleavage was monitored by HPLC (See the schematic diagram in  FIG. 16 .) To carry out these experiments, the concentration of β-secretase required for cleavage of both substrates was determined as shown in  FIG. 17 . For substantial cleavage of ST6Gal1 substrate, 420 nM of the enzyme was required. Therefore, 420 nM of β-secretase was used for the following experiment.  
      Inhibitory activity of inhibitors, APPsw inhibitor, c-Sub MΔN2C1 and commercially available β-secretase inhibitor, on each β-secretase substrate was observed as shown in  FIG. 18 . When a 25-fold increase in the amount of the inhibitor was added, about 60% of Sub M cleavage was blocked and only 25% of ST6Gal1 peptide cleavage was blocked. However, the commercially available β-secretase inhibitor was equally effective in blocking both substrates. Therefore, the inventive APP inhibitor is specific for APP.  
     Example 14  
     Peptides that Bind to the γ-Secretase Cleavage Site of APP  
      Decamer peptide sequences that contain the cleavage site of APP by γ-secretase was used. The sequence is as follow: GVVIATVIVI (SEQ ID NO:8). γ-secretase cleaves the peptide bond between A and T and releases the following cleavage products: GVVIA (SEQ ID NO:41) and TVIVI (SEQ ID NO:42) ( FIG. 19 ).  
      As described in Example 1, we designed two peptides based on the hydropathic complementary approach. The anti-sense sequences were deduced from the mRNA sequences corresponding to the above-described decamer substrate peptides. Genetic codes were derived from the antisense RNA by reading the sequences either in 5′→3′ or 3′→5′ directions. As shown in  FIG. 19 , the following decamer peptide sequences were obtained: DDDHCRYDNT (γCh1(5′→3′), SEQ ID NO:31) and PQQYRCHRQR (γCh2 (3′→5′), SEQ ID NO:9). These peptides are collectively called γ complementary peptides. For γCh2, since there are two stop codons according to the genetic code, arginine has been inserted for the stop codons.  
     Example 15  
     γ-Secretase Activity Assay  
      Since γ-secretase is composed of four components, cloning of γ-secretase gene is impossible. Therefore, cell extracts were used as γ-secretase source. To obtain the cell extracts, after cell lysis with extraction buffer, the lysate was centrifuged at 10,000×g for 1 minute. Afterward, 2× reaction buffer and fluorogenic substrate was mixed and added to the cell lysate. Then, this mixture was incubated at 37° C. and γ-secretase activity was detected at excitation 335 to 355 nm and emission 495 to 510 nm.  
     Example 16  
     γ-Secretase Cleavage Activity  
      In order to choose a cell line with the highest γ-secretase cleavage activity, four different cell lines were tested according to the assay method described in Example 15. As shown in  FIG. 20 , all types of cell lines exhibited time dependent γ-secretase activity. Among these, N2a, which is mouse neuroblastoma, showed the highest activity and was chosen as the source of γ-secretase.  
     Example 17  
     Effect of Complementary Peptides on γ-Secretase Activity  
      γ-Secretase activity assay was performed on membrane fractions of N2a cells in the presence of several complementary peptides. After 12.5 μM fluorogenic substrate and 200 μM each of the complementary peptides were preincubated for 1 hour, γ-secretase was added to the mixture. In the course of time, the fluorogenic substrate was cleaved by the γ-secretase. As shown in  FIG. 21 , rCh2 (3′→5′) had the highest inhibitory effect (about 80%) while the other tested complementary peptides inhibited γ-secretase activity only slightly.  
     Example 18  
     Cell Based γ-Secretase Assay  
      In order to test these peptides in the cell for their inhibitory effect on 7-secretase cleavage, a cell based assay was developed. After KEK293 APP cells were cultured in 6 well culture plates with 90% confluency, the cells were treated for 9 hours with N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester (DAPT) (Dovey HF et al, J. Neurochemistry 2001; 76:173-181), which is a known γ-secretase inhibitor, and complementary peptides. The cells in each well were lysed and these lysates were separated with 15% tris-tricine gel. Western analysis was performed with R1 antibody as primary antibody and goat anti-rabbit-HRP as secondary antibody.  
      As shown in  FIG. 22 , DAPT inhibits γ-secretase activity very effectively (lane 1). The resultant α-CTF (C-terminal fragment), which is a product of α-secretase cleavage cannot be cleaved by γ-secretase and instead accumulates in the membrane. However, complementary peptides tested do not have inhibitory effect when compared with control. These results indicate that the complementary peptides cannot be transported into the cell across the membrane. Complementary peptides in the γ-secretase inhibition experiments, rCh2 (3′→5′) coupled with polyarginine is tested for translocation across the cell membrane and inhibitory activity in the cells.  
     Example 19  
     Alanine Scanning of c-Sub MΔC1N3 (CIQIHF)  
      Each position of CIQIHF (SEQ ID NO:29) was replaced with Alanine to identify the amino acid that is important for the peptide&#39;s inhibitory activity. Replacement with Alanine would presumably reduce the inhibitory activity of the original peptide sequence. The inhibitor activity of the original peptide and the peptides replaced with Alanine at each position were determined as described in Example 4 above. The results show that the amino acids at the first (C), second (I), fourth (I) and sixth (F) positions are significant for the inhibitory activity of CIQIHF.  
      All of the references cited herein are incorporated by reference in their entirety.  
      Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention specifically described herein. Such equivalents are intended to be encompassed in the scope of the claims.