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Compounds That Bind APP and Inhibit Aβ Processing in Vitro Suggest a Novel Approach to Alzheimer Disease Therapeutics

2005; Elsevier BV; Volume: 280; Issue: 18 Linguagem: Inglês

10.1074/jbc.m414331200

1083-351X

Amy S. Espeseth, Min Xu, Qian Huang, Craig A. Coburn, Kristen L. Jones, Marc Ferrer, Paul Zuck, Berta Strulovici, Eric A. Price, Guoxin Wu, Abigail Wolfe, Janet Lineberger, Mohinder K. Sardana, Katherine Tugusheva, Beth Pietrak, Ming‐Chih Crouthamel, Ming‐Tain Lai, Elizabeth Chen Dodson, Renzo Bazzo, Xiao‐Ping Shi, Adam J. Simon, Yue‐Ming Li, Daria J. Hazuda,

Computational Drug Discovery Methods

Extracellular deposits of aggregated amyloid-β (Aβ) peptides are a hallmark of Alzheimer disease; thus, inhibition of Aβ production and/or aggregation is an appealing strategy to thwart the onset and progression of this disease. The release of Aβ requires processing of the amyloid precursor protein (APP) by both β- and γ-secretase. Using an assay that incorporates full-length recombinant APP as a substrate for β-secretase (BACE), we have identified a series of compounds that inhibit APP processing, but do not affect the cleavage of peptide substrates by BACE1. These molecules also inhibit the processing of APP and Aβ by BACE2 and selectively inhibit the production of Aβ42 species by γ-secretase in assays using CTF99. The compounds bind directly to APP, likely within the Aβ domain, and therefore, unlike previously described inhibitors of the secretase enzymes, their mechanism of action is mediated through APP. These studies demonstrate that APP binding agents can affect its processing through multiple pathways, providing proof of concept for novel strategies aimed at selectively modulating Aβ production. Extracellular deposits of aggregated amyloid-β (Aβ) peptides are a hallmark of Alzheimer disease; thus, inhibition of Aβ production and/or aggregation is an appealing strategy to thwart the onset and progression of this disease. The release of Aβ requires processing of the amyloid precursor protein (APP) by both β- and γ-secretase. Using an assay that incorporates full-length recombinant APP as a substrate for β-secretase (BACE), we have identified a series of compounds that inhibit APP processing, but do not affect the cleavage of peptide substrates by BACE1. These molecules also inhibit the processing of APP and Aβ by BACE2 and selectively inhibit the production of Aβ42 species by γ-secretase in assays using CTF99. The compounds bind directly to APP, likely within the Aβ domain, and therefore, unlike previously described inhibitors of the secretase enzymes, their mechanism of action is mediated through APP. These studies demonstrate that APP binding agents can affect its processing through multiple pathways, providing proof of concept for novel strategies aimed at selectively modulating Aβ production. Alzheimer disease (AD) 1The abbreviations used are: AD, Alzheimer disease; APP, amyloid precursor protein; BACE, β-amyloid cleaving enzyme; Aβ, amyloid β; CTF99, 99-amino acid C-terminal fragment; MBP, maltose-binding protein; NSAIDs, nonsteroidal anti-inflammatory drugs; sAPPβNF, N-terminal cleavage product of APP with the enhanced BACE substrate NFEV; FL, full-length. is the leading cause of senile dementia. AD currently affects 4.5 million people in the United States and is anticipated to increase to more than 13 million by the year 2050 (1Hebert L.E. Scherr P.A. Bienias J.L. Bennett D.A. Evans D.A. Arch. Neurol. 2003; 60: 1119-1122Crossref PubMed Scopus (1943) Google Scholar, 2Hebert L.E. Beckett L.A. Scherr P.A. Evans D.A. Alzheimer Dis. Assoc. Disord. 2001; 15: 169-173Crossref PubMed Scopus (385) Google Scholar). Histopathologically, AD is characterized by the prevalence of senile plaques, as well as neocortical atrophy, neuronal and synaptic loss, and the formation of neuritic tangles of the microtubule-associated protein tau (3Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5170) Google Scholar). Senile plaques consist predominantly of fibrils of amyloid β (Aβ), a 39–43-amino acid peptide generated by proteolysis of the amyloid precursor protein (APP) (4Hardy J.A. Higgins G.A. Science. 1992; 256: 184-185Crossref PubMed Scopus (5120) Google Scholar). Production of Aβ has been linked genetically to AD. Recent studies (5Lacor P.N. Buniel M.C. Chang L. Fernandez S.J. Gong Y. Viola K.L. Lambert M.P. Velasco P.T. Bigio E.H. Finch C.E. Krafft G.A. Klein W.L. J. Neurosci. 2004; 24: 10191-10200Crossref PubMed Scopus (849) Google Scholar, 6Klein W.L. Krafft G.A. Finch C.E. Trends Neurosci. 2001; 24: 219-224Abstract Full Text Full Text PDF PubMed Scopus (916) Google Scholar, 7Hardy J. Selkoe D. Science. 2002; 297: 353-356Crossref PubMed Scopus (11023) Google Scholar) have shown that Aβ multimerizes into neurotoxic oligomers that target the synapse, leading to disruption of long term potentiaton, cell death, and memory loss. These oligomers then coalesce into fibrils, ultimately forming the plaques originally used to characterize the disease (5Lacor P.N. Buniel M.C. Chang L. Fernandez S.J. Gong Y. Viola K.L. Lambert M.P. Velasco P.T. Bigio E.H. Finch C.E. Krafft G.A. Klein W.L. J. Neurosci. 2004; 24: 10191-10200Crossref PubMed Scopus (849) Google Scholar, 6Klein W.L. Krafft G.A. Finch C.E. Trends Neurosci. 2001; 24: 219-224Abstract Full Text Full Text PDF PubMed Scopus (916) Google Scholar, 7Hardy J. Selkoe D. Science. 2002; 297: 353-356Crossref PubMed Scopus (11023) Google Scholar). To produce Aβ, APP, a type I single-transmembrane glycoprotein, is sequentially cleaved by two aspartyl proteases, β- and γ-secretase. The initial cleavage is mediated by β-secretase (BACE1) on the luminal side of the endosomal membrane or at the cell surface (8Chyung J.H. Selkoe D.J. J. Biol. Chem. 2003; 278: 51035-51043Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). BACE1 cleavage generates a secreted N-terminal product (sAPPβ) and a transmembrane N-terminal product (CTF99). Processing of CTF99 within the transmembrane domain by the γ-secretase enzyme complex generates the C-terminal end of Aβ and results in its release (9Sisodia S.S. St George-Hyslop P.H. Nat. Rev. Neurosci. 2002; 3: 281-290Crossref PubMed Scopus (487) Google Scholar). The γ-secretase enzyme is known to cleave CTF99 at any of several discrete positions, generating Aβ peptides that range in length from 38 to 42 amino acids. Although Aβ40 is the predominant species produced both in vitro and in vivo, Aβ42 is the more aggregenic species and the major component of amyloid plaques. The release of Aβ42 and subsequent aggregation into insoluble fibrils are believed to lead to the formation of the dense plaques characteristic of AD (10Walsh D.M. Klyubin I. Fadeeva J.V. Rowan M.J. Selkoe D.J. Biochem. Soc. Trans. 2002; 30: 552-557Crossref PubMed Scopus (455) Google Scholar). Drug discovery efforts aimed at preventing the accumulation of Aβ have been directed toward inhibition of Aβ production and prevention of Aβ aggregation (11Dingwall C. J. Clin. Investig. 2001; 108: 1243-1246Crossref PubMed Scopus (34) Google Scholar, 12Wolfe M.S. Nat. Rev. Drug. Discov. 2002; 1: 859-866Crossref PubMed Scopus (179) Google Scholar) as well as enhancing Aβ clearance (13Findeis M.A. Curr. Top. Med. Chem. 2002; 2: 417-423Crossref PubMed Scopus (81) Google Scholar, 14Findeis M.A. Biochim. Biophys. Acta. 2000; 1502: 76-84Crossref PubMed Scopus (182) Google Scholar, 15Weggen S. Eriksen J.L. Das P. Sagi S.A. Wang R. Pietrzik C.U. Findlay K.A. Smith T.E. Murphy M.P. Bulter T. Kang D.E. Marquez-Sterling N. Golde T.E. Koo E.H. Nature. 2001; 414: 212-216Crossref PubMed Scopus (1333) Google Scholar). Preventing Aβ production has focused on targeting the enzymes involved in processing APP. Transition state-based inhibitors of BACE1 have been described with in vitro activity, as well as activity in cell culture (16Hu J. Cwi C.L. Smiley D.L. Timm D. Erickson J.A. McGee J.E. Yang H.C. Mendel D. May P.C. Shapiro M. McCarthy J.R. Bioorg. Med. Chem. Lett. 2003; 13: 4335-4339Crossref PubMed Scopus (44) Google Scholar, 17Hom R.K. Fang L.Y. Mamo S. Tung J.S. Guinn A.C. Walker D.E. Davis D.L. Gailunas A.F. Thorsett E.D. Sinha S. Knops J.E. Jewett N.E. Anderson J.P. John V. J. Med. Chem. 2003; 46: 1799-1802Crossref PubMed Scopus (98) Google Scholar, 18Tung J.S. Davis D.L. Anderson J.P. Walker D.E. Mamo S. Jewett N. Hom R.K. Sinha S. Thorsett E.D. John V. J. Med. Chem. 2002; 45: 259-262Crossref PubMed Scopus (128) Google Scholar, 19Ghosh A.K. Bilcer G. Harwood C. Kawahama R. Shin D. Hussain K.A. Hong L. Loy J.A. Nguyen C. Koelsch G. Ermolieff J. Tang J. J. Med. Chem. 2001; 44: 2865-2868Crossref PubMed Scopus (236) Google Scholar, 20Hom R.K. Gailunas A.F. Mamo S. Fang L.Y. Tung J.S. Walker D.E. Davis D. Thorsett E.D. Jewett N.E. Moon J.B. John V. J. Med. Chem. 2004; 47: 158-164Crossref PubMed Scopus (112) Google Scholar, 21Chen S.H. Lamar J. Guo D. Kohn T. Yang H.C. McGee J. Timm D. Erickson J. Yip Y. May P. McCarthy J. Bioorg. Med. Chem. Lett. 2004; 14: 245-250Crossref PubMed Scopus (43) Google Scholar, 22Lamar J. Hu J. Bueno A.B. Yang H.C. Guo D. Copp J.D. McGee J. Gitter B. Timm D. May P. McCarthy J. Chen S.H. Bioorg. Med. Chem. Lett. 2004; 14: 239-243Crossref PubMed Scopus (49) Google Scholar, 23Brady S.F. Singh S. Crouthamel M. Holloway M.K. Coburn C.A. Garsky V. Bogusky M. Pennington M.W. Vacca J.P. Hazuda D. Lai M-T. Bioorg. Med. Chem. 2004; 14: 601-604Crossref Scopus (60) Google Scholar, 24Coburn C.A. Stachel S.J. Li Y. Rush D.M. Steele T. Chen Dodson E. Holloway M.K. Xu M. Huang Q. Lai M. Dimuzio-Mower J.M. Crouthamel M.C. Shi X. Sardana V. Chen Z. Munshi S. Kuo L. Makara G. Annis D.A. Tadikonda P.K. Nash H.M. Vacca J.P. J. Med. Chem. 2004; 47: 6117-6119Crossref PubMed Scopus (123) Google Scholar, 25Stachel S.J. Coburn C.A. Steele T. Jones K.G. Loutzenkiser E.F. Gregro A.R. Rajapakse H.A. Lai M.T. Crouthamel M.C. Xu M. Tugusheva K. Lineberger J.E. Pietrak B.L. Espeseth A.S. Shi X. Chen-Dodson E. Holloway M.K. Munshi S.K. Simon A.J. Kuo L.C. Vacca J.P. J. Med. Chem. 2004; 47: 6447-6450Crossref PubMed Scopus (278) Google Scholar), but data on in vivo efficacy is limited. Although numerous γ-secretase inhibitors have been shown to block Aβ processing in vitro and in vivo (26Lewis H.D. Perez Revuelta B.I. Nadin A. Neduvelil J.G. Harrison T. Pollack S.J. Shearman M.S. Biochemistry. 2003; 42: 7580-7586Crossref PubMed Scopus (70) Google Scholar, 27Shearman M.S. Beher D. Clarke E.E. Lewis H.D. Harrison T. Hunt P. Nadin A. Smith A.L. Stevenson G. Castro J.L. Biochemistry. 2000; 39: 8698-8704Crossref PubMed Scopus (367) Google Scholar, 28Li Y.M. Xu M. Lai M.T. Huang Q. Castro J.L. DiMuzio-Mower J. Harrison T. Lellis C. Nadin A. Neduvelil J.G. Register R.B. Sardana M.K. Shearman M.S. Smith A.L. Shi X.P. Yin K.C. Shafer J.A. Gardell S.J. Nature. 2000; 405: 689-694Crossref PubMed Scopus (865) Google Scholar, 29Churcher I. Williams S. Kerrad S. Harrison T. Castro J.L. Shearman M.S. Lewis H.D. Clarke E.E. Wrigley J.D. Beher D. Tang Y.S. Liu W. J. Med. Chem. 2003; 46: 2275-2278Crossref PubMed Scopus (71) Google Scholar, 30Dovey H.F. John V. Anderson J.P. Chen L.Z. de Saint Andrieu P. Fang L.Y. Freedman S.B. Folmer B. Goldbach E. Holsztynska E.J. Hu K.L. Johnson-Wood K.L. Kennedy S.L. Kholodenko D. Knops J.E. Latimer L.H. Lee M. Liao Z. Lieberburg I.M. Motter R.N. Mutter L.C. Nietz J. Quinn K.P. Sacchi K.L. Seubert P.A. Shopp G.M. Thorsett E.D. Tung J.S. Wu J. Yang S. Yin C.T. Schenk D.B. May P.C. Altstiel L.D. Bender M.H. Boggs L.N. Britton T.C. Clemens J.C. Czilli D.L. Dieckman-McGinty D.K. Droste J.J. Fuson K.S. Gitter B.D. Hyslop P.A. Johnstone E.M. Li W.Y. Little S.P. Mabry T.E. Miller F.D. Audia J.E. J. Neurochem. 2001; 76: 173-181S. A. P.Crossref PubMed Scopus (797) Google Scholar), γ-secretase plays an essential role in the processing of other, disparate targets, and mechanism-based toxicity is a potential concern (31Kopan R. Goate A. Genes Dev. 2000; 14: 2799-2806Crossref PubMed Scopus (189) Google Scholar, 32Searfoss G.H. Jordan W.H. Calligaro D.O. Galbreath E.J. Schirtzinger L.M. Berridge B.R. Gao H. Higgins M.A. May P.C. Ryan T.P. J. Biol. Chem. 2003; 278: 46107-46116Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 33Hadland B.K. Manley N.R. Su D. Longmore G.D. Moore C.L. Wolfe M.S. Schroeter E.H. Kopan R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7487-7491Crossref PubMed Scopus (189) Google Scholar, 34De Strooper B. Annaert W. Cupers P. Saftig P. Craessaerts K. Mumm J.S. Schroeter E.H. Schrijvers V. Wolfe M.S. Ray W.J. Goate A. Kopan R. Nature. 1999; 398: 518-522Crossref PubMed Scopus (1800) Google Scholar). In contrast, molecules known as γ-secretase modulators selectively inhibit γ-secretase processing of APP at the 42 cleavage site and may have a reduced effect on processing other substrates; however, this has not been extensively studied to date. Herein we describe a novel series of benzofuran-containing compounds that inhibit APP processing by a previously undescribed mechanism of action. Although the compounds were initially identified in a search for BACE1 inhibitors, they inhibit BACE1, BACE2, and γ-secretase-mediated cleavage of APP by binding APP within the Aβ domain of the protein. Some of these inhibitors preferentially affect γ-secretase processing at the 42 cleavage site; however, their mechanism of action is distinct from γ-secretase modulators. These studies provide evidence that targeting APP may present opportunities to identify small molecules that affect APP processing and provide a novel strategy to develop chemotherapeutic agents for AD. Materials—BACE1 protein was produced from a recombinant baculovirus expression system, and BACE2 protein was produced from HEK293T cells as described in Refs. 35Shi X.P. Chen E. Yin K.C. Na S. Garsky V.M. Lai M.T. Li Y.M. Platchek M. Register R.B. Sardana M.K. Tang M.J. Thiebeau J. Wood T. Shafer J.A. Gardell S.J. J. Biol. Chem. 2001; 276: 10366-10373Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar and 36Shi X-P. Wu G.-X. Tugusheva K. Register R.B. Chen-Dodson E. Price E. Sardana M. Lucka A. Bruce J.E. Hazuda D.J. Neurobiol. Aging. 2002; 23: 180Google Scholar. γ-Secretase was purified from HeLa S3 cells as described by Li et al. (37Li Y.M. Lai M.T. Xu M. Huang Q. DiMuzio-Mower J. Sardana M.K. Shi X.P. Yin K.C. Shafer J.A. Gardell S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6138-6143Crossref PubMed Scopus (499) Google Scholar). In Vitro Secretase Assays—BACE1 and BACE2 cleavage of peptide substrates and Aβ was carried out as described in Refs. 23Brady S.F. Singh S. Crouthamel M. Holloway M.K. Coburn C.A. Garsky V. Bogusky M. Pennington M.W. Vacca J.P. Hazuda D. Lai M-T. Bioorg. Med. Chem. 2004; 14: 601-604Crossref Scopus (60) Google Scholar, 36Shi X-P. Wu G.-X. Tugusheva K. Register R.B. Chen-Dodson E. Price E. Sardana M. Lucka A. Bruce J.E. Hazuda D.J. Neurobiol. Aging. 2002; 23: 180Google Scholar, and 38Shi X.P. Tugusheva K. Bruce J.E. Lucka A. Wu G.X. Chen-Dodson E. Price E. Li Y. Xu M. Huang Q. Sardana M.K. Hazuda D.J. J. Biol. Chem. 2003; 278: 21286-21294Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar. Mass spectrometric detection of BACE2 cleavage of Aβ by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (Ciphergen Biosystems) was done as described in Ref. 38Shi X.P. Tugusheva K. Bruce J.E. Lucka A. Wu G.X. Chen-Dodson E. Price E. Li Y. Xu M. Huang Q. Sardana M.K. Hazuda D.J. J. Biol. Chem. 2003; 278: 21286-21294Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar. Peptides assayed for BACE1 and BACE2 cleavage are described in the legend to Fig. 3. To assay cleavage of APPFL, purified biotinylated maltose-binding protein (MBP)-fused, bacterially expressed, full-length APP containing an enhanced BACE1 cleavage sequence was incubated with compound and BACE1 protein for 1 h at 37°Cor BACE2 protein for 30 min at 37 °C. The cleavage product was detected using streptavidin-conjugated donor AlphaScreen beads (20 μg/ml, PerkinElmer Life Sciences) and polyclonal anti-sAPPβNF (39Shi X-P. Tugusheva K. Bruce J.E. Lucka A. Chen Dodson E. Hu B. Wu G. Price E. Register R.B. Lineberger J. Gates A.T. Miller R. Tang M. Espeseth A. Kahana J. Wolfe A. Crouthamel M. Sankaranarayanan S. Simon A. Chen L. Lai M.-T. Pietrak B. Dimuzio-Mower J. Li Y. Xu M. Huang Q. Garsky V. Sardana M.K. Hazuda D.J. JAD. 2005; 7: 2Crossref Scopus (36) Google Scholar) bound to protein A-coated acceptor AlphaScreen beads (20 μg/ml, PerkinElmer Life Sciences) in 0.1% bovine serum albumin in phosphate-buffered saline. Plates were incubated overnight in the dark and then read using an Alpha-Fusion HT instrument (PerkinElmer Life Sciences). γ-Secretase was assayed as described previously (37Li Y.M. Lai M.T. Xu M. Huang Q. DiMuzio-Mower J. Sardana M.K. Shi X.P. Yin K.C. Shafer J.A. Gardell S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6138-6143Crossref PubMed Scopus (499) Google Scholar, 40Xu M. Lai M.T. Huang Q. DiMuzio-Mower J. Castro J.L. Harrison T. Nadin A. Neduvelil J.G. Shearman M.S. Shafer J.A. Gardell S.J. Li Y.M. Neurobiol. Aging. 2002; 23: 1023-1030Crossref PubMed Scopus (32) Google Scholar). Mass spectrometric analysis of CTF99 cleavage was carried out using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (Ciphergen Biosystems, Palo Alto, CA) as described by Li et al. (37Li Y.M. Lai M.T. Xu M. Huang Q. DiMuzio-Mower J. Sardana M.K. Shi X.P. Yin K.C. Shafer J.A. Gardell S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6138-6143Crossref PubMed Scopus (499) Google Scholar). Cell-based Assay for γ-Secretase Activity—H4 neuroglioma cells were stably transfected with the SPβA4CTF expression vector (27Shearman M.S. Beher D. Clarke E.E. Lewis H.D. Harrison T. Hunt P. Nadin A. Smith A.L. Stevenson G. Castro J.L. Biochemistry. 2000; 39: 8698-8704Crossref PubMed Scopus (367) Google Scholar). The cells were treated with compound for 16–20 h, and conditioned media were harvested. Aβ X-40 and X-42 in conditioned media were assayed via electrochemiluminescence using an Origen 1.5 Analyzer (Igen), essentially as described in Ref. 37Li Y.M. Lai M.T. Xu M. Huang Q. DiMuzio-Mower J. Sardana M.K. Shi X.P. Yin K.C. Shafer J.A. Gardell S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6138-6143Crossref PubMed Scopus (499) Google Scholar, except using biotinylated 6E10 (1 μg/ml) (Signet Laboratories) and ruthenylated G2–10 (0.375 μg/ml) (41Ida N. Hartmann T. Pantel J. Schroder J. Zerfass R. Forstl H. Sandbrink R. Masters C.L. Beyreuther K. J. Biol. Chem. 1996; 271: 22908-22914Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar) for detection of Aβ X-40 and biotinylated 6E10 (1 μg/ml) and ruthenylated G2–11 (0.375 μg/ml) (41Ida N. Hartmann T. Pantel J. Schroder J. Zerfass R. Forstl H. Sandbrink R. Masters C.L. Beyreuther K. J. Biol. Chem. 1996; 271: 22908-22914Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar) for detection of Aβ X-42. Surface Plasmon Resonance Binding Assay—Sensorgrams for binding of compounds 1–3 onto MBP-APPFL were generated using a Biacore S51 instrument. Equal amounts of recombinant MBP-APPFL and MBP-sAPPβNF proteins were immobilized on Spot 1 or Spot 2 in Flow cell 1 of a Sensor Chip SA (Biacore), respectively. The compounds were dissolved in 5% Me2SO in phosphate-buffered saline. The sensorgrams were recorded at a flow rate of 30 μl/min at 30 °C. The compound was injected for 30 s. Following compound injection, 5% Me2SO/phosphate-buffered saline buffer was injected over the chip for another 30 s. A solvent correction cycle was run for each sample. Specific binding to MBP-APPFL was calculated as binding to MBP-APPFL – MBP-sAPPβNF binding. Identification of a Novel Group of Inhibitors That Inhibit BACE1 Cleavage of Full-length APP but Not Peptide Substrates—In the process of performing a high throughput screen for BACE1 inhibitors using a purified recombinant full-length APP substrate, we identified the novel benzofuran-containing compound 1 shown in Table I. The activity of compound 1 and a panel of related analogs (compounds 2–5, Table I) was evaluated using an AlphaScreen assay for BACE1 cleavage of APP (see "Experimental Procedures"). This assay is a homogeneous proximity-based method that uses donor beads binding both cleaved and uncleaved APP and acceptor beads coated with an antibody that binds only the cleaved APP product. Excitation of the donor bead triggers the release of singlet oxygen, which then stimulates the emission of light in the acceptor. With the exception of compound 4, all four analogs inhibited BACE1 cleavage of APP with IC50 values ranging from 11 to 50 μm (Table I). The effect of these compounds on BACE1 cleavage of APP was confirmed by Western blot analysis of the reaction products. For compounds 1–4 the relative potencies for APP processing inhibition did not vary significantly between the AlphaScreen and Western assays (Fig. 1, A and C, and data not shown). However, compound 5 did not inhibit APP cleavage when assayed via Western blot, and subsequent analyses suggested compound 5 may have disrupted detection in the AlphaScreen assay by triggering the precipitation of substrate (data not shown). The specificity of this class of compounds for inhibition of APP processing was established by reviewing data from over 70 assays run at ≥5 μm that included compound 2 (the most potent compound). Compound 2 was not active in a wide variety of binding, cell-based, or enzymatic activity screens (data not shown).Table IBenzofuran-containing compounds block BACE1 cleavage of APPFL but not a peptide-based substrate (P5/P5′) Open table in a new tab Fig. 1Inhibition of BACE1 cleavage of full-length APP or a β-site P5/P5′ peptide by compounds 1 and 2 or Statine-Val. Compound 1 (A) and Statine-Val (B) were compared for their ability to inhibit cleavage of a full-length APP substrate ○ or a β-site P5-P5′ peptide (□). Enzymatic activity is shown relative to Me2SO-treated BACE1 cleavage. C, Western analysis of BACE1 cleavage of APPFL in the presence of a titration of compound 2 and densitometric analysis of the percent cleaved. The Western blot of the cleavage reaction was probed with a sAPPβNF-neo-epitope antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Although compounds 1–4 reproducibly blocked BACE1 cleavage of APP, the benzofuran analogs did not inhibit BACE1 processing of peptide substrates, such as P5-P5′ (Table I, Fig. 1A), and extended substrates encompassing P21-P10′ (see Fig. 3). In contrast, a well characterized transition state mimic inhibitor of BACE1, Statine-Val, blocked BACE1 cleavage of the peptide and APPFL substrates (Fig. 1B) with comparable potencies, suggesting the inability of the benzofurans to block cleavage of the P5-P5′ peptide may be related to a functionally distinct mechanism of action. Compounds 1–4 Exhibit Substrate-dependent Activity on BACE1 and -2 as Well as γ-Secretase—To further examine the substrate-dependent activity of these inhibitors, we evaluated their effect on the closely related protease BACE2. Although BACE2 also processes APP at the β-site, it preferentially cleaves APP at two sites positioned at 19 and 20 amino acids within Aβ (42Farzan M. Schnitzler C.E. Vasilieva N. Leung D. Choe H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9712-9717Crossref PubMed Scopus (347) Google Scholar), as well as at position 34 (38Shi X.P. Tugusheva K. Bruce J.E. Lucka A. Wu G.X. Chen-Dodson E. Price E. Li Y. Xu M. Huang Q. Sardana M.K. Hazuda D.J. J. Biol. Chem. 2003; 278: 21286-21294Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Inhibition of BACE2 cleavage at the β-site was first tested using full-length APP and the P5-P5′ peptide as described for BACE1 (Table II). Compounds 1–3 inhibited BACE2 cleavage of full-length APP with IC50 values that were similar to inhibition of BACE1 cleavage. Compound 4 weakly inhibited full-length APP cleavage, with an IC50 value of ∼100 μm. Again, these compounds did not block BACE2 cleavage of the P5-P5′ peptide, although the transition state analog inhibitor was effective.Table IIBACE 2 inhibition with APP, peptide substrate (P9–P5′ 14-mer), or AβCompoundAPP IC50P9/P5′ IC50Aβ 19/20 (+/-)Aβ 34 (+/-)μmμm120>100Enhanced++++29>100++++++34>100-++++4∼100>100+++++5NDaND, not determined.NDNDNDa ND, not determined. Open table in a new tab The effect of compounds 1–4 on BACE2 cleavage of Aβ was tested by incubating Aβ (1–40) with BACE2 in the presence or absence of a concentration of 100 μm for each compound and resolving the cleavage products by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Cleavage of Aβ (1–40) at position 34 was reduced by all four compounds (Fig. 2 and Table II). Surprisingly, the effect on BACE2 processing at positions 19 and 20 in Aβ was compound-specific. Compound 3 had little effect on BACE2 cleavage at positions 19 and 20, whereas compounds 2 and 4 blocked cleavage by ∼50%. In contrast, compound 1 substantially increased BACE2 cleavage at these positions (Fig. 2 and Table II). The differential effects of the benzofurans on BACE1 and BACE2 activity with respect to peptide versus protein substrates and BACE2 activity with regard to processing distinct sites within Aβ is novel for compounds that inhibit BACE1/2 activity. A potential explanation for the distinctive substrate dependence exhibited by benzofurans 1–4 is that the compounds block cleavage of APP and Aβ by binding to and interfering with the substrate. In this case, the compounds might also block γ-secretase cleavage of substrates containing Aβ. Compounds 1–5 were therefore evaluated for inhibition of γ-secretase cleavage of both CTF99 and a 28-mer peptide encompassing the C-terminal domain of Aβ extending from residues 28 through 55 of CTF99 (Table III and Fig. 3). Compounds 1–3 inhibited γ-secretase cleavage of CTF99 at the X-40 position with IC50 values similar to those observed for inhibition of BACE1 and BACE2 cleavage of APP. (Cleavage of CTF99 at the X-42 position was too low to be detected). Compounds 1–3 also inhibited γ-secretase cleavage of the 28-mer peptide substrate, and the IC50 values obtained in assays using the 28-mer peptide were comparable with those obtained with CTF99. Compound 4 inhibited γ-secretase cleavage of the CTF99 but did not inhibit cleavage of the peptide substrate. Compound 5 did not have an effect on cleavage of CTF99 or the peptide substrate.Table IIIγ-Secretase inhibition with CTF99 or a 28-mer peptide substrateCompoundCTF99 IC5028-mer IC50μmμm1NDaND, not determined.1022911387450>2005>33>100a ND, not determined. Open table in a new tab Compounds 1–3 Bind APP within the Aβ Domain—The benzofurans 1–4 inhibited processing of a variety of related APP substrates by structurally distinct aspartyl proteases. Substrates in which cleavage was affected had in common the C-terminal domain of Aβ. Taken together, the data suggest these compounds may interfere with processing by interacting with the substrates rather than the enzymes, and this interaction is likely mediated through the C-terminal portion of the Aβ domain (Fig. 3). Although we attempted to explore the potential for interaction with APP using a variety of biophysical methods, such as NMR and nitrocellulose filter binding, the limited solubility of these compounds was problematic for many solution-based methods (data not shown). We therefore used surface plasmon resonance to assess binding to APP using the Biacore S51 instrument. Biotinylated APPFL and biotinylated sAPPβ were immobilized onto a streptavidin-coated sensor chip. Compounds 1–3 were analyzed at various concentrations, and the binding response at each concentration was monitored for both proteins. Representative data for binding to APPFL are shown for compound 2 in Fig. 4. For compound 2, binding to APPFL was observed to occur in a dose-dependent manner (Fig. 4A), whereas no interaction was observed between the BACE inhibitor, Statine-Val, and APP (Fig. 4B). Compound 2 did not bind to sAPPβ, consistent with the results from enzyme assays, which suggest binding to APPFL occurs within the Aβ domain (data not shown). Finally, the Kd determined in this analysis (43 μm) was comparable with the IC50 values obtained for this compound for inhibition of APP processing by BACE1 and γ-secretase (9–29 μm) (Fig. 4B). APP binding was also observed with compounds 1 and 3 (data not shown). The Benzofuran Analogs Inhibit CTF99 Cleavage in Cell Culture—Compounds 1–4 were tested in cell culture for their effects on APP processing by BACE1 and γ-secretase (39Shi X-P. Tugusheva K. Bruce J.E. Lucka A. Chen Dodson E. Hu B. Wu G. Price E. Register R.B. Lineberger J. Gates A.T. Miller R. Tang M. Espeseth A. Kahana J. Wolfe A. Crouthamel M. Sankaranarayanan S. Simon A. Chen L. Lai M.-T. Pietrak B. Dimuzio-Mower J. Li Y. Xu M. Huang Q. Garsky V. Sardana M.K. Hazuda D.J. JAD. 2005; 7: 2Crossref Scopus (36) Google Scholar). Although none of the compounds inhibited BACE1 cleavage of APP, all four analogs inhibited γ-secretase cleavage of exogenously expressed CTF99. Interestingly, compounds 2 and 3, similar to a number of NSAIDs, had position-specific effects on γ-secretase cleavage of CTF99 (Fig. 5B). As with the γ-secretase modulators, production of the Aβ X-42 species of amyloid was more sensitive to treatment with the benzofuran compounds than was the Aβ X-40 species (Fig. 5). We have characterized a series of benzofuran analogs that inhibit secretase-mediated APP processing by a novel mechanism. These molecules inhibit BACE1 and BACE2 processing of full-length APP but not β-site peptide substrates. In addition, they inhibit BACE2 processing of Aβ and γ-secretase processing of CTF99 with comparable efficacy. Although BACE1 and γ-secretase are both aspartyl proteases, they do not share structural similarity, and to date, there have been no other reports of compounds that block both β- and γ-secretase-mediated proteolysis in vitro. Inhibition of both BACE1/2 and γ-secretase cleavage, along with the substrate specificity associated with compound activity and the demonstration of binding to APP by surface plasmon resonance, suggest these molecules block APP processing, not by interacting with the processing enzymes, but by binding to the substrate. These results validate a new mechanism to interfere with APP processing and suggest a novel approach to AD therapeutics. These compounds were able to disrupt γ-secretase processing of both APP and CTF99 in vitro, but in cells, they prevented only γ-secretase cleavage of CTF99, an apparent contradiction. The physical properties of the compounds used in this study made it unlikely that they would cross the plasma membrane, so one possible explanation is that γ-secretase cleavage of CTF99 occurs at the plasma membrane, whereas full-length APP is internalized upon interaction with BACE1 and is cleaved by both BACE1 and γ-secretase within the endosome (43Kinoshita A. Fukumoto H. Shah T. Whelan C.M. Irizarry M.C. Hyman B.T. J. Cell Sci. 2003; 116: 3339-3346Crossref PubMed Scopus (226) Google Scholar). γ-Secretase activity at the plasma membrane, including interactions between γ-secretase and CTF99, has been described previously, supporting this assertion (8Chyung J.H. Selkoe D.J. J. Biol. Chem. 2003; 278: 51035-51043Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 34De Strooper B. Annaert W. Cupers P. Saftig P. Craessaerts K. Mumm J.S. Schroeter E.H. Schrijvers V. Wolfe M.S. Ray W.J. Goate A. Kopan R. Nature. 1999; 398: 518-522Crossref PubMed Scopus (1800) Google Scholar, 45Chyung J.H. Raper D.M. Selkoe D. J. Biol. Chem. 2005; 280: 4383-4392Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 47Ray W.J. Yao M. Mumm J.S. Schroeter E.H. Saftig P. Wolfe M.S. Selkoe D. Kopan R. Goate A. J. Biol. Chem. 1999; 274: 36801-36807Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 48Tarassishin L. Yin Y.I. Bassit B. Li Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17050-17055Crossref PubMed Scopus (94) Google Scholar). Although other studies carried out with cell impenetrant γ-secretase inhibitors have suggested that γ-secretase cleavage of Notch occurs at the cell surface, while cleavage of CTF99 occurs in the endosomal compartment; however, differences in cell lines and in expression conditions between these assays and those used in this study may have resulted in differences in CTF99 trafficking that could account for these differences (48Tarassishin L. Yin Y.I. Bassit B. Li Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17050-17055Crossref PubMed Scopus (94) Google Scholar). Indirect evidence implicating binding to APP through the C-terminal portion of the Aβ includes the following observations: (i) compounds 1–4 blocked BACE1 and BACE2 cleavage of APP at the β-site but had no effect on the processing of β-site peptide substrates, (ii) the compounds inhibited BACE2 cleavage of Aβ at position 34 but exhibited variable efficacy at the 19/20 site, and finally, (iii) γ-secretase cleavage of a peptide beginning at Aβ position 28 was inhibited. Although these data allow one to infer that binding occurs within the Aβ domain proximal to the C-terminal end, it is interesting to note that these compounds inhibit cleavage of APP by BACE1 and -2 at the N terminus of Aβ. Therefore, although these effects on substrate processing may be the result of direct steric interference, it is also possible that binding may induce conformational changes and prevent processing indirectly. The latter could explain the enhancement of processing by BACE2 observed at specific cleavage sites. Interestingly, structurally distinct benzofuran analogs that dissociate fibrillar Aβ have been described previously (49Howlett D.R. Perry A.E. Godfrey F. Swatton J.E. Jennings K.H. Spitzfaden C. Wadsworth H. Wood S.J. Markwell R.E. Biochem. J. 1999; 340: 283-289Crossref PubMed Scopus (190) Google Scholar, 50Ono M. Kung M.P. Hou C. Kung H.F. Nucl. Med. Biol. 2002; 29: 633-642Crossref PubMed Scopus (139) Google Scholar), and the benzofuran motif in both series may play a critical role in binding to Aβ. The C terminus of Aβ is known to be involved in aggregation, thus the observation that benzofuran analogs can affect Aβ aggregation is consistent with our results, suggesting that benzofurans 1–4 interact with Aβ within this region. For compound 2, the Kd determined for binding to full-length APP by surface plasmon resonance was similar to the IC50 value obtained for BACE1 and γ-secretase cleavage of APP, as well as BACE2 cleavage of Aβ. Taken together, these results suggest the structure of the Aβ domain may not differ significantly between APP and the processed peptide. Intriguingly, the benzofuran-containing compounds, although inhibiting cleavage at the β-site and at Aβ position 34, also behaved like cleavage modulators. At positions 19 and 20 of Aβ, compound 1 enhanced BACE2 cleavage, although reducing BACE2 cleavage at position 34. In addition, when assayed for their effects on γ-secretase cleavage of CTF99 in cells, compounds 2 and 3 inhibited cleavage at position 42, although having a decreased effect on cleavage at position 40. This differential effect on γ-secretase cleavage of CTF99 in cells is similar to the reported effects of NSAIDs on γ-secretase cleavage of APP, both in mice and in cells. Several mechanisms for the effect of NSAIDs on Aβ secretion have been proposed. The γ-secretase modulatory effects of NSAIDs have been ascribed as an indirect effect because of their inhibition of Rho (51Zhou Y. Su Y. Li B. Liu F. Ryder J.W. Wu X. Gonzalez-DeWhitt P.A. Gelfanova V. Hale J.E. May P.C. Paul S.M. Ni B. Sastre M. Dewachter I. Landreth G.E. Willson T.M. Klockgether T. van Leuven F. Heneka M.T. Yan Q. Zhang J. Liu H. Babu-Khan S. Vassar R. Biere A.L. Citron M. Landreth G. Martyn C. Eriksen J.L. Sagi S.A. Smith T.E. Weggen S. Das P. McLendon D.C. Ozols V.V. Jessing K.W. Zavitz K.H. Koo E.H. Golde T.E. Pietrzik C.U. Ozols V. Fauq A. Eriksen J. Takahashi Y. Hayashi I. Tominari Y. Rikimaru K. Morohashi Y. Kan T. Natsugari H. Fukuyama T. Tomita T. Iwatsubo T. Morihara T. Chu T. Ubeda O. Beech W. Cole G.M. Science. 2003; 302: 1215-1217Crossref PubMed Scopus (309) Google Scholar). However, NSAIDs directly block γ-secretase activity in cell extracts and compete with transition state inhibitors for binding to γ-secretase, suggesting a noncompetitive allosteric binding mode (46Beher D. Clarke E.E. Wrigley J.D. Martin A.C. Nadin A. Churcher I. Shearman M.S. J. Biol. Chem. 2004; 279: 43419-43426Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 52Weggen S. Eriksen J.L. Sagi S.A. Pietrzik C.U. Ozols V. Fauq A. Golde T.E. Koo E.H. J. Biol. Chem. 2003; 278: 31831-31837Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 53Takahashi Y. Hayashi I. Tominari Y. Rikimaru K. Morohashi Y. Kan T. Natsugari H. Fukuyama T. Tomita T. Iwatsubo T. J. Biol. Chem. 2003; 278: 18664-18670Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Therefore, it appears unlikely that the benzofurans described in this manuscript modulate APP cleavage via the same mechanism as the NSAIDs. However, NSAIDs have also been shown to bind Aβ fibrils (44Agdeppa E.D. Kepe V. Petri A. Satyamurthy N. Liu J. Huang S.C. Small G.W. Cole G.M. Barrio J.R. Neuroscience. 2003; 117: 723-730Crossref PubMed Scopus (168) Google Scholar). Although it is possible that an additional mechanism for the γ-secretase modulatory effects of NSAIDs is mediated by the APP CTF, the structure of Aβ fibrils is distinct from the APPFL, CTF99, and soluble Aβ substrates of β- and γ-secretase. Our results demonstrate that compounds that bind APP can inhibit its processing by each of the three different enzymes responsible for its metabolism in vivo. This represents a novel mechanism for inhibition of Aβ generation. Although the higher molar concentration of substrate relative to enzyme suggests that small molecule inhibitors are more effective when directed to the enzyme component of the pathway, given the potential for mechanism-based toxicity associated with γ-secretase inhibitors and the overall difficulties in generating BACE1 inhibitors with in vivo efficacy, targeting APP may provide an alternative approach for AD drug discovery efforts.