The Amyloid-β Rise and γ-Secretase Inhibitor Potency Depend on the Level of Substrate Expression

The amyloid-β (Aβ) peptide, which likely plays a key role in Alzheimer disease, is derived from the amyloid-β precursor protein (APP) through consecutive proteolytic cleavages by β-site APP-cleaving enzyme and γ-secretase. Unexpectedly γ-secretase inhibitors can increase the secretion of Aβ peptides under some circumstances. This “Aβ rise” phenomenon, the same inhibitor causing an increase in Aβ at low concentrations but inhibition at higher concentrations, has been widely observed. Here we show that the Aβ rise depends on the β-secretase-derived C-terminal fragment of APP (βCTF) or C99 levels with low levels causing rises. In contrast, the N-terminally truncated form of Aβ, known as “p3,” formed by α-secretase cleavage, did not exhibit a rise. In addition to the Aβ rise, low βCTF or C99 expression decreased γ-secretase inhibitor potency. This “potency shift” may be explained by the relatively high enzyme to substrate ratio under conditions of low substrate because increased concentrations of inhibitor would be necessary to affect substrate turnover. Consistent with this hypothesis, γ-secretase inhibitor radioligand occupancy studies showed that a high level of occupancy was correlated with inhibition of Aβ under conditions of low substrate expression. The Aβ rise was also observed in rat brain after dosing with the γ-secretase inhibitor BMS-299897. The Aβ rise and potency shift are therefore relevant factors in the development of γ-secretase inhibitors and can be evaluated using appropriate choices of animal and cell culture models. Hypothetical mechanisms for the Aβ rise, including the “incomplete processing” and endocytic models, are discussed. The amyloid-β (Aβ) peptide, which likely plays a key role in Alzheimer disease, is derived from the amyloid-β precursor protein (APP) through consecutive proteolytic cleavages by β-site APP-cleaving enzyme and γ-secretase. Unexpectedly γ-secretase inhibitors can increase the secretion of Aβ peptides under some circumstances. This “Aβ rise” phenomenon, the same inhibitor causing an increase in Aβ at low concentrations but inhibition at higher concentrations, has been widely observed. Here we show that the Aβ rise depends on the β-secretase-derived C-terminal fragment of APP (βCTF) or C99 levels with low levels causing rises. In contrast, the N-terminally truncated form of Aβ, known as “p3,” formed by α-secretase cleavage, did not exhibit a rise. In addition to the Aβ rise, low βCTF or C99 expression decreased γ-secretase inhibitor potency. This “potency shift” may be explained by the relatively high enzyme to substrate ratio under conditions of low substrate because increased concentrations of inhibitor would be necessary to affect substrate turnover. Consistent with this hypothesis, γ-secretase inhibitor radioligand occupancy studies showed that a high level of occupancy was correlated with inhibition of Aβ under conditions of low substrate expression. The Aβ rise was also observed in rat brain after dosing with the γ-secretase inhibitor BMS-299897. The Aβ rise and potency shift are therefore relevant factors in the development of γ-secretase inhibitors and can be evaluated using appropriate choices of animal and cell culture models. Hypothetical mechanisms for the Aβ rise, including the “incomplete processing” and endocytic models, are discussed. Evidence suggests that the amyloid-β (Aβ) 9The abbreviations used are:Aβamyloid-βAPPamyloid-β precursor proteinBACEβ-site APP-cleaving enzymeBMSBristol-Myers SquibbCHAPSO3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonateβCTFβ-secretase-derived C-terminal fragment of APPDAPTN-[N-(3,5-difluorophenacetyl)-l-alanyl]-(S)-phenylglycine t-butyl esterDMEMDulbecco's modified Eagle's mediumDMSOdimethyl sulfoxideELISAenzyme-linked immunosorbent assayFADfamilial Alzheimer diseaseHEKhuman embryonic kidneyHEKswHEK293 cells stably transfected with APP Swedish variantHEKwtHEK293 cells stably transfected with APP wild typeLC/MSliquid chromatography mass spectroscopyMES2-(N-morpholino)ethanesulfonic acidPBSphosphate-buffered salineαCTFα-secretase-derived C-terminal fragment of APPHPLChigh pressure liquid chromatographyarbsarbitrary unitsbis-tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolE/Senzyme to substrate. peptide plays a key role in Alzheimer disease. Aβ is generated by proteolytic processing of the amyloid-β precursor protein (APP) through consecutive cleavages by the β-site APP-cleaving enzyme (BACE) and γ-secretase. APP is cleaved by BACE to form a β-secretase-derived C-terminal fragment of APP (βCTF), which undergoes further cleavage by γ-secretase to form Aβ. In addition, APP is cleaved by α-secretase to form αCTF, which undergoes γ-secretase cleavage to produce an N-terminally truncated form of Aβ known as “p3.” Using the conventional amino acid numbering of Aβ, BACE cleavage leads to Aβ peptides with N-terminal ends at positions 1 and 11, whereas α-secretase leads to p3 peptides with an N-terminal end at position 17. Cleavage of βCTF and αCTF by γ-secretase produces a mixture of different C termini in the resulting Aβ and p3 peptides. For example, the predominant γ-secretase cleavage of βCTFs at position 40 produces Aβ-(1–40) and Aβ-(11–40), whereas other γ-secretase cleavage sites produce a range of less abundant Aβ peptides, such as the disease-associated Aβ-(1–42) (1Hardy J. Selkoe D.J. Science. 2002; 297: 353-356Crossref PubMed Scopus (11360) Google Scholar, 2Selkoe D.J. Schenk D. Annu. Rev. Pharmacol. Toxicol. 2003; 43: 545-584Crossref PubMed Scopus (754) Google Scholar). amyloid-β amyloid-β precursor protein β-site APP-cleaving enzyme Bristol-Myers Squibb 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate β-secretase-derived C-terminal fragment of APP N-[N-(3,5-difluorophenacetyl)-l-alanyl]-(S)-phenylglycine t-butyl ester Dulbecco's modified Eagle's medium dimethyl sulfoxide enzyme-linked immunosorbent assay familial Alzheimer disease human embryonic kidney HEK293 cells stably transfected with APP Swedish variant HEK293 cells stably transfected with APP wild type liquid chromatography mass spectroscopy 2-(N-morpholino)ethanesulfonic acid phosphate-buffered saline α-secretase-derived C-terminal fragment of APP high pressure liquid chromatography arbitrary units 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol enzyme to substrate. Although γ-secretase cleavage can be fully inhibited in cell-based assays, some inhibitors cause an increase in the amount of Aβ at subinhibitory concentrations. This “Aβ rise” phenomenon, the same inhibitor causing an increase in Aβ at low concentrations but inhibition at higher concentrations, has been observed frequently (3Citron M. Diehl T.S. Gordon Selkoe D.J. PubMed Scopus Google Scholar, D. M. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar, M. Diehl T.S. Selkoe D.J. J. PubMed Scopus Google Scholar, Selkoe D.J. PubMed Scopus Google Scholar, Diehl T.S. Selkoe D.J. PubMed Scopus Google Scholar, J. Pharmacol. PubMed Scopus Google Scholar, M. J. M. D. PubMed Scopus Google Scholar). For peptide some studies a rise that was for (3Citron M. Diehl T.S. Gordon Selkoe D.J. PubMed Scopus Google Scholar, D. M. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, PubMed Scopus Google whereas other studies a rise also for in addition to PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar). studies also as to the to the on some the an on γ-secretase PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google whereas a on γ-secretase (3Citron M. Diehl T.S. Gordon Selkoe D.J. PubMed Scopus Google Scholar, D. M. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). In the Aβ rise was in a of peptide on γ-secretase PubMed Scopus Google Scholar). that γ-secretase can the Aβ rise from studies with which are for γ-secretase and which cause a rise in in cell culture M. Diehl T.S. Selkoe D.J. J. PubMed Scopus Google Scholar, Selkoe D.J. PubMed Scopus Google Scholar, Diehl T.S. Selkoe D.J. PubMed Scopus Google and in PubMed Scopus Google Scholar). a rise in Aβ, as as in to γ-secretase inhibitors has been observed in in the of J. Pharmacol. PubMed Scopus Google and in M. J. M. D. PubMed Scopus Google Scholar). the of the Aβ rise has not been and the conditions for this not been Here we show that a rise in Aβ can be observed in cell with γ-secretase inhibitors and that the key is a low level of βCTF or C99 expression. In addition, low substrate expression a shift in inhibitor potency that was of the Aβ rise. also show that increased Aβ can in the brain γ-secretase inhibitor dosing in the of γ-secretase inhibitors to cause the of the in the the Aβ rise is a relevant in the development of γ-secretase inhibitors for and conditions that exhibit the Aβ rise can be in cell culture models. and D. J. PubMed Scopus Google D. M. J. J. Schenk D. J. PubMed Scopus Google and M. J. PubMed Scopus Google from M. J. Pharmacol. PubMed Scopus Google M. J. PubMed Scopus Google and PubMed Scopus Google been is a and is an The radioligand is an γ-secretase inhibitor that to the inhibitor D. J. 2003; PubMed Scopus Google Scholar, D. J. PubMed Scopus Google Scholar). This radioligand is on for high in embryonic cell is from embryonic cells which γ-secretase D. PubMed Scopus Google Scholar, D. M. PubMed Scopus Google Scholar). and cells stably transfected with APP wild type or Swedish variant derived as D.J. PubMed Scopus Google Scholar, M. Selkoe D.J. J. 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