3,4-Fused cyclohexyl sulfones as γ-secretase inhibitors
Duncan ShawJonathan D. BestKevin DinnellAlan NadinMark S. ShearmanChristine PattisonJames PeacheyMichael ReillyBrian J. WilliamsJonathan D.J. WrigleyTimothy Harrison
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Sulfone
Structure–activity relationship
Amyloid-β peptide (AβP) that accumulates in the Alzheimer's diseased brain is derived from proteolytic processing of the amyloid precursor protein (APP) by means of β- and γ-secretases. The β-secretase APP cleaving enzyme (BACE), which generates the N terminus of AβP, has become a target of intense research aimed at blocking the enzyme activity, thus reducing AβP and, subsequently, plaque formation. The search for specific inhibitors of β-secretase activity as a possible treatment for Alzheimer's disease intensified with the discovery that BACE may be involved in processing other non-APP substrates. The presence of the APP–BACE complex in early endosomes highlights the cell surface as a potential therapeutic target, suggesting that interference in APP–BACE interaction at the cell surface may affect amyloid-β production. We present here a unique approach to inhibit AβP production by means of antibodies against the β-secretase cleavage site of APP. These antibodies were found to bind human APP overexpressed by CHO cells, and the formed immunocomplex was visualized in the early endosomes. Indeed, blocking of the β-secretase site by these antibodies interfered with BACE activity and inhibited both intracellular and extracellular AβP formation in these cells.
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The presenilin 1 (PS1) and presenilin 2 (PS2) proteins are necessary for proteolytic cleavage of the amyloid precursor protein (APP) within its transmembrane domain. One of these cleavage events (termed γ-secretase) generates the C-terminal end of the Aβ-peptide by proteolysis near residue 710 or 712 of APP770. Another event (termed γ-like or ε-secretase cleavage) cleaves near residue 721 at ∼2–5 residues inside the cytoplasmic membrane boundary to generate a series of stable, C-terminal APP fragments. This latter cleavage is analogous to S3-cleavage of Notch. We report here that specific mutations in the N terminus, loop, or C terminus of PS1 all increase the production of Aβ42 but cause inhibition of both ε-secretase cleavage of APP and S3-cleavage of Notch. These data support the hypothesis that ε-cleavage of APP and S3-cleavage of Notch are similar events. They also argue that, although both the γ-site and the ε-site cleavage of APP are presenilin-dependent, they are likely to be independent catalytic events.
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Most gene mutations associated with Alzheimer’s disease point to the metabolism of amyloid precursor protein as a potential cause. The β- and γ-secretases are two executioners of amyloid precursor protein processing resulting in amyloid-β. Significant progress has been made in the selective inhibition of both proteases, regardless of structural information for γ-secretase. Several peptidic and nonpeptidic leads were identified for both targets.
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Neurotoxic amyloid β-peptides are thought to be a causative agent of Alzheimer's disease in humans. The production of amyloid β-peptides from amyloid precursor protein (APP) could be diminished by enhancing α-processing; however, the physical interactions between APP and α-secretases are not well understood. In this study, we employed super-resolution light microscopy to examine in cell-free plasma membranes the abundance and association of APP and α-secretases ADAM10 (a disintegrin and metalloproteinase) and ADAM17. We found that both secretase molecules localize similarly closely to APP (within ≤50 nm). However, when cross-linking APP with antibodies directed against the GFP tag of APP, in confocal microscopy, we observed that only ADAM10 coaggregated with APP. Furthermore, we mapped the involved protein domain by using APP variants with an exchanged transmembrane segment or lacking cytoplasmic/extracellular domains. We identified that the transmembrane domain of APP is required for association with α-secretases and, as analyzed by Western blot, for α-processing. We propose that the transmembrane domain of APP interacts either directly or indirectly with ADAM10, but not with ADAM17, explaining the dominant role of ADAM10 in α-processing of APP. Further understanding of this interaction may facilitate the development of a therapeutic strategy based on promoting APP cleavage by α-secretases.
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Alzheimer-disease-associated β-amyloid (Aβ) is produced by sequential endoproteolysis of β-amyloid protein precursor (βAPP): the extracellular portion is shed by cleavage in the juxtamembrane region by β-amyloid-cleaving enzyme (BACE)/β-secretase, after which it is cleaved by presenilin (PS)/γ-secretase near the middle of the transmembrane domain. Thus, inhibition of either of the secretases reduces Aβ generation and is a fundamental strategy for the development of drugs to prevent Alzheimer disease. However, it is not clear how small compounds reduce Aβ production without inhibition of the secretases. Such compounds are expected to avoid some of the side effects of secretase inhibitors. Here, we report that destruxin E (Dx-E), a natural cyclic hexadepsipeptide, reduces Aβ generation without affecting BACE or PS/γ-secretase activity. In agreement with this, Dx-E did not inhibit Notch signaling. We found that Dx-E decreases colocalization of BACE1 and βAPP, which reduces β-cleavage of βAPP. Therefore, the data demonstrate that Dx-E represents a novel Aβ-reducing process which could have fewer side effects than secretase inhibitors.
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Neuritic plaques in the brain are a major neuropathological hallmark of Alzheimer’s disease. They are formed by the deposition and aggregation of extracellular amyloid-β protein (Aβ). Aβ is derived from the sequential cleavage of amyloid-β precursor protein (APP) by β-secretase and γ-secretase. β-Site APP cleaving enzyme 1 (BACE1) functions as the primary, if not sole, β-secretase in vivo and is essential for Aβ production. Regulation of APP processing is a major focus of research into AD pathogenesis. The trafficking systems of APP and its cleavage enzymes are complex. Transporting APP and secretases into the same subcellular organelles facilitates their interaction and favors APP processing. The role of APP and BACE1 trafficking in the amyloidgenic pathway and the underlying mechanisms for Aβ production are discussed in this review. In addition, the distinct mechanisms of amino- and carboxy-terminal Aβ generation are reviewed.
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Abstract Cleavage of amyloid‐β precursor protein ( APP ) at the Asp1 β‐secretase site of the amyloid‐β protein (Aβ) domain by β‐site Aβ precursor protein‐cleaving enzyme 1 ( BACE 1) is required for the generation of Aβ, a central component of neuritic plaques in the Alzheimer's disease ( AD ) brain. In this study, we found that Aβ Glu11 is the major β‐secretase site for cleavage of APP by BACE 1 to generate soluble secreted APP ( sAPP β) 606 and the C‐terminal membrane‐bound fragment ( CTF )β product C89. Cleavage of C89 by γ‐secretase resulted in truncated Aβ generation in a non‐amyloidogenic pathway. A familial AD ‐associated Swedish APP mutation adjacent to Aβ Asp1 shifted the major APP β‐secretase cleavage site from Aβ Glu11 to Asp1, resulting in significant increases in sAPP β596 and CTF β C99 generation and the C99/89 ratio, in turn leading to increased Aβ production in cultured cells in vitro and transgenic AD model mouse brains in vivo . Furthermore, increased BACE 1 expression facilitated APP being processed by the β‐secretase processing pathway rather than the α‐secretase pathway, leading to more Aβ production. Our results suggest that potentiating BACE 1 cleavage of APP at both the Asp1 and Glu11 sites, or shifting the cleavage from the Glu11 site to the Asp1 site, could result in increased Aβ production and facilitate neuritic plaque formation. Our study provides new insights into how alteration of BACE 1 expression and β‐secretase cleavage site selection could contribute to Alzheimer pathogenesis and the pharmaceutical potential of modulating BACE1 expression and its cleavage site selection.
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Alzheimer's disease (AD) is a common neurodegenerative disease whose prevalence increases with age. An increasing number of findings suggest that abnormalities in the metabolism of amyloid precursor protein (APP), a single transmembrane aspartic protein that is cleaved by β- and γ-secretases to produce β-amyloid protein (Aβ), are a major pathological feature of AD. In recent years, a large number of studies have been conducted on the APP processing pathways and the role of secretion. This paper provides a summary of the involvement of secretases in the processing of APP and the potential drug targets that could provide new directions for AD therapy.
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β-Amyloid peptide (Aβ) is a principal component of parenchymal amyloid deposits in Alzheimer's disease. Aβ is derived from amyloid precursor protein (APP) by proteolytic cleavage. APP is subject to N- and O-glycosylation and potential tyrosine sulfation, following protein synthesis, and is then thought to be cleaved in an intracellular secretory pathway after or during these post-translational modifications. Studies utilizing agents that affect a series of steps in the protein secretory pathway have identified the possible intracellular sites of APP cleavage and Aβ generation within the protein secretory pathway. In the present study, using cells with normal protein metabolism, but expressing mutant APP with defectiveO-glycosylation, we demonstrated that the majority of APP cleavage by α-, β-, and γ-secretases occurs afterO-glycosylation. Cells expressing the mutant APP noticeably decreased the generation of the intracellular APP carboxyl-terminal fragment (αAPPCOOH), a product of α-secretase, and both Aβ40 and Aβ42 in medium, a product of β- and γ-secretases. Furthermore, we found that the cells accumulate the mutant APP in intracellular reticular compartments such as the endoplasmic reticulum. Agents that could ambiguously affect the function of specific intracellular organelles and that may be toxic were not used. The present results indicate that APP is cleaved by α-, β-, and γ-secretases in step(s) during the transport of APP through Golgi complex, where O-glycosylation occurs, or in compartments subsequent to trans-Golgi of the APP secretory pathway. β-Amyloid peptide (Aβ) is a principal component of parenchymal amyloid deposits in Alzheimer's disease. Aβ is derived from amyloid precursor protein (APP) by proteolytic cleavage. APP is subject to N- and O-glycosylation and potential tyrosine sulfation, following protein synthesis, and is then thought to be cleaved in an intracellular secretory pathway after or during these post-translational modifications. Studies utilizing agents that affect a series of steps in the protein secretory pathway have identified the possible intracellular sites of APP cleavage and Aβ generation within the protein secretory pathway. In the present study, using cells with normal protein metabolism, but expressing mutant APP with defectiveO-glycosylation, we demonstrated that the majority of APP cleavage by α-, β-, and γ-secretases occurs afterO-glycosylation. Cells expressing the mutant APP noticeably decreased the generation of the intracellular APP carboxyl-terminal fragment (αAPPCOOH), a product of α-secretase, and both Aβ40 and Aβ42 in medium, a product of β- and γ-secretases. Furthermore, we found that the cells accumulate the mutant APP in intracellular reticular compartments such as the endoplasmic reticulum. Agents that could ambiguously affect the function of specific intracellular organelles and that may be toxic were not used. The present results indicate that APP is cleaved by α-, β-, and γ-secretases in step(s) during the transport of APP through Golgi complex, where O-glycosylation occurs, or in compartments subsequent to trans-Golgi of the APP secretory pathway. Alzheimer's disease (AD) 1The abbreviations used are: AD, Alzheimer's disease; Aβ, β-amyloid; APP, amyloid precursor protein; αAPPCOOH, α-secretase cleaved intracellular APP carboxyl-terminal fragment; ConA, concanavalin A; ELISA, enzyme-linked immunosorbent assay; ER, endoplasmic reticulum; FAD, familial Alzheimer's disease; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; WGA, wheat germ agglutinin; wt, wild type; imAPP, immature APP; mAPP, mature APP; APPmut, mutant APP; PBS, phosphate-buffered saline. is characterized by the presence of parenchymal and cerebrovascular β-amyloid (Aβ) deposits (1Glenner G. Wong C. Biochem. Biophys. Res. Commun. 1984; 122: 885-890Crossref Scopus (4238) Google Scholar, 2Masters C.L. Simms G. Weinmann N.A. Multhaup G. McDonald B.L. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4245-4249Crossref PubMed Scopus (3679) Google Scholar). Aβ is a 39–43-amino acid peptide that is derived from Alzheimer's amyloid precursor protein (APP). The generation of Aβ is thought to be one of the major events of AD pathogenesis (reviewed in Refs. 3Codell B. Annu. Rev. Pharmacol. Toxicol. 1994; 34: 69-89Crossref PubMed Google Scholar and 4Selkoe D.J. Annu. Rev. Neurosci. 1994; 17: 489-517Crossref PubMed Scopus (829) Google Scholar). APP is an integral membrane protein with a receptor-like structure, existing in several isoforms which, in many tissues, arise by alternative splicing of a single gene (5Goldgaber D. Lerman M.I. McBride O.W. Saffiotti U. Gajdusek D.C. Science. 1987; 235: 877-880Crossref PubMed Scopus (1026) Google Scholar, 6Kang J. Lemaire H.G. Unterbeck A.J. Salbaum J.M. Master C.L. Grzeschik K.H. Multhaup G. Beyreuther K. Muller-Hill B. Nature. 1987; 325: 733-736Crossref PubMed Scopus (3956) Google Scholar, 7Robakis N.K. Ramakrishna N. Wolfe G. Wisniewski H.M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4190-4194Crossref PubMed Scopus (505) Google Scholar, 8Tanzi R.E. Gusella J.F. Watkins P.C. Brus G.A.P. St George-Hyslop P. Van Keuren M.L. Patterson D. Pagan S. Kurnit D.M. Neve R.L. Science. 1987; 235: 880-884Crossref PubMed Scopus (1224) Google Scholar, 9Tanzi R.E. McClatchey A.I. Lamperti E.D. Villa-Komaroff L. Gusella J.F. Neve R.L. Nature. 1988; 331: 528-530Crossref PubMed Scopus (872) Google Scholar, 10Ponte P. Gonzales-De Whitt P. Schilling J. Miller J. Hsu D. Greenberg B. Davis K. Wallace W. Lieberburg I. Fuller F. Cordell B. Nature. 1988; 331: 525-527Crossref PubMed Scopus (858) Google Scholar, 11Kitaguchi N. Takahashi Y. Tokushima Y. Shiojiri S. Ito H. Nature. 1988; 331: 530-532Crossref PubMed Scopus (886) Google Scholar, 12De Sauvage F. Octave J.-N. Science. 1989; 245: 651-653Crossref PubMed Scopus (160) Google Scholar). APP is subject to post-translational modification such as glycosylation, sulfation, and phosphorylation during transit through the intracellular protein secretory pathway (13Weidemann A. Konig G. Bunke D. Fischer P. Salbaum J.M. Master C.L. Beyreuther K. Cell. 1989; 57: 115-126Abstract Full Text PDF PubMed Scopus (1038) Google Scholar, 14Oltersdorf T. Ward P.J. Henriksson T. Beattie E.C. Neve R. Lieberburg I. Fritz L.C. J. Biol. Chem. 1990; 265: 4492-4497Abstract Full Text PDF PubMed Google Scholar, 15Påhlsson P. Shakin-Eshleman S.H. Spitalnik S.L. Biochem. Biophys. Res. Commun. 1992; 189: 1667-1673Crossref PubMed Scopus (71) Google Scholar, 16Knops J. Gandy S. Greengard P. Lieberburg I. Sinha S. Biochem. Biophys. Res. Commun. 1993; 197: 380-385Crossref PubMed Scopus (22) Google Scholar, 17Hung A.Y. Selkoe D.J. EMBO J. 1994; 13: 534-542Crossref PubMed Scopus (112) Google Scholar, 18Suzuki T. Oishi M. Marshak D.R. Czernik A.J. Nairn A.C. Greengard P. EMBO J. 1994; 13: 1114-1122Crossref PubMed Scopus (212) Google Scholar, 19Graebert K.S. Popp G.M. Kehlw T. Herzog V. Eur. J. Cell Biol. 1995; 66: 39-46PubMed Google Scholar, 20Påhlsson P. Spitalnik S.L. Arch. Biochem. Biophys. 1996; 331: 177-186Crossref PubMed Scopus (55) Google Scholar, 21Oishi M. Nairn A.C. Czernik A.J. Lim G.S. Isohara T. Gandy S.E. Greengard P. Suzuki T. Mol. Med. 1997; 3: 111-123Crossref PubMed Google Scholar, 22Walter J. Capell A. Hung A.Y. Langen H. Schnölzer M. Thinakaran G. Sisodia S.S. Selkoe D.J. Haass C. J. Biol. Chem. 1997; 272: 1896-1903Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). APP isoforms exist as immature (imAPP, N-glycosylated) and mature (mAPP, N- and O-glycosylated, tyrosyl-sulfated) species. The imAPP localizes in the ER and cis-Golgi, and the mAPP localizes in compartments following trans-Golgi and on the plasma membrane. The molecular mechanism(s) and cellular compartment(s) involved in APP cleavage and Aβ production have yet to be fully resolved. Studies using agents (i.e. brefeldin A and monensin) or studies with treatments (i.e. cell culture at low temperature) that interfere with secretory metabolic steps (23Sambamurti K. Shioi J.P. Pappolla M.A. Robakis N.K. J. Neurosci. Res. 1992; 33: 319-329Crossref PubMed Scopus (134) Google Scholar, 24De Strooper B. Umans L. Van Leuven F. Van Den Berghe H. J. Cell Biol. 1993; 121: 295-304Crossref PubMed Scopus (140) Google Scholar, 25Haass C. Hung A.Y. Schlossmacher M.G. Teplow D.B. Selkoe D.J. J. Biol. Chem. 1993; 268: 3021-3024Abstract Full Text PDF PubMed Google Scholar, 26Kuentzel S.L. Ali S.M. Altman R.A. Greenberg B.D. Raub T.J. Biochem. J. 1993; 295: 367-378Crossref PubMed Scopus (119) Google Scholar, 27Refolo L.M. Sambamurti K. Efthimiopoulos S. Pappolla M.A. Robakis N.K. J. Neurosci. Res. 1995; 40: 694-706Crossref PubMed Scopus (52) Google Scholar, 28Thinakaran G. Teplow D.B. Siman R. Greenberg B. Sisodia S.S. J. Biol. Chem. 1996; 271: 9390-9397Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar) suggest that APP cleavage by α-secretase occurs in a secretory step in late Golgi. Although recent reports indicate that the ER is the site for generation of Aβ42 but not Aβ40 in the neuron (29Hartmann T. Bieger S.C. Brühl B. Tienari P.J. Ida N. Allsop D. Roberts G.W. Masters C.L. Dotti C.G. Unsicker K. Beyreuther K. Nat. Med. 1997; 3: 1016-1020Crossref PubMed Scopus (646) Google Scholar, 30Cook D.G. Forman M.S. Sung J.C. Leight S. Kolson D.L. Iwatsubo T. Lee V.M.-Y. Doms R.W. Nat. Med. 1997; 3: 1021-1023Crossref PubMed Scopus (430) Google Scholar), Aβ in studies using agents that interfere with pH gradients (i.e.chloroquine and ammonium chloride) is believed to be generated in acidic compartments such as endosomes and/or late Golgi (31Haass C. Schlossmacher M.G. Hung A.Y. Vigo-Pelfrey C. Mellon A. Ostaszewski B.L. Lieberburg I. Koo E.H. Schenk D. Teplow D.B. Selkoe D.J. Nature. 1992; 359: 322-325Crossref PubMed Scopus (1765) Google Scholar, 32Shoji M. Golde T.E. Ghiso J. Cheung T.T. Estus S. Shaffer L.M. Cai X.-D. McKay D.M. Tintner R. Frangione B. Younkin S.G. Science. 1992; 258: 126-129Crossref PubMed Scopus (1327) Google Scholar, 33Koo E.H. Squazzo S.L. J. Biol. Chem. 1994; 269: 17386-17389Abstract Full Text PDF PubMed Google Scholar). However, these procedures are toxic, and it is possible that these agents interfere with intracellular protein metabolism through nonspecific and unpredictable mechanisms. To identify potential intracellular compartments involved in the cleavage of APP by secretases without utilizing toxic metabolic inhibitors, we prepared cells expressing mutant APP (APPmut) which is not subject to O-glycosylation. In such cells, all other intracellular protein metabolism is thought to be normal. Taking advantage of the property of the cells expressing APPmut, we examined the processing of APP in healthy cells. Cells expressing the APPmut noticeably decreased the generation of the carboxyl-terminal fragment of APP (αAPPCOOH), a product of cleavage by α-secretase, and also failed to generate Aβ40 and Aβ42, products of cleavage by both β- and γ-secretases. The present study shows that, without utilizing metabolic agents which nonspecifically interfere with protein degradation and secretion, APP is cleaved after, or possibly during, maturation (O-glycosylation). These results indicate that APP cleavage occurs in compartment(s) subsequent to trans-Golgi of the protein secretory pathway or possibly during the transport of APP through Golgi complex, where O-glycosylation occurs (34Danphy W.G. Rothman J.E. Cell. 1985; 42: 13-21Abstract Full Text PDF PubMed Scopus (281) Google Scholar). Generation of Aβ42 in the ER (29Hartmann T. Bieger S.C. Brühl B. Tienari P.J. Ida N. Allsop D. Roberts G.W. Masters C.L. Dotti C.G. Unsicker K. Beyreuther K. Nat. Med. 1997; 3: 1016-1020Crossref PubMed Scopus (646) Google Scholar, 30Cook D.G. Forman M.S. Sung J.C. Leight S. Kolson D.L. Iwatsubo T. Lee V.M.-Y. Doms R.W. Nat. Med. 1997; 3: 1021-1023Crossref PubMed Scopus (430) Google Scholar) may be a neuron-specific and/or a minor event.
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Ectodomain
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α,β and γ secretases are three kinds of key enzymes in processing and metabolizing amyloid precursor protein(APP).Their selective activation of the α secretase or inhibition of β and γ secretase will help reduce the production of β-amyloid protein(Aβ),and may become one of the effective targets to treating AD.This paper aims to make a review on the research development of relevant factors influencing the activity and expression of 3 APP secretases and their targeted intervention in pharmaceutical studies.
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Amyloid (mycology)
Gamma secretase
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