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 G. Biere A.L. Seubert P. Selkoe D.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13170-13175Crossref PubMed Scopus (279) Google Scholar, 4Klafki H.-W. Abramowski D. Swoboda R. Paganetti P.A. Staufenbiel M. J. Biol. Chem. 1996; 271: 28655-28659Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 5Durkin J.T. Murthy S. Husten E.J. Trusko S.P. Savage M.J. Rotella D.P. Greenberg B.D. Siman R. J. Biol. Chem. 1999; 274: 20499-20504Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 6Zhang L. Song L. Terracina G. Liu Y. Pramanik B. Parker E. Biochemistry. 2001; 40: 5049-5055Crossref PubMed Scopus (92) Google Scholar, 7Yamazaki T. Haass C. Saido T.C. Omura S. Ihara Y. Biochemistry. 1997; 36: 8377-8383Crossref PubMed Scopus (56) Google Scholar, 8Higaki J.N. Chakravarty S. Bryant C.M. Cowart L.R. Harden P. Scardina J.M. Mavunkel B. Luedtke J.R. Cordell B. J. Med. Chem. 1999; 42: 3889-3898Crossref PubMed Scopus (47) Google Scholar, 9Zhang L. Song L. Parker E.M. J. Biol. Chem. 1999; 274: 8966-8972Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 10Dong Y. Tan J. Cui M.-Z. Zhao G. Mao G. Singh N. Xu X. FASEB J. 2006; 20: 331-333Crossref PubMed Scopus (23) Google Scholar, 11Wolfe M.S. Citron M. Diehl T.S. Xia W. Donkor I.O. Selkoe D.J. J. Med. Chem. 1998; 41: 6-9Crossref PubMed Scopus (220) Google Scholar, 12Wolfe M.S. Xia W. Moore C.L. Leatherwood D.D. Ostaszewsky B.L. Rahmati T. Donkor I.O. Selkoe D.J. Biochemistry. 1999; 38: 4720-4727Crossref PubMed Scopus (309) Google Scholar, 13Moore C.L. Diehl T.S. Selkoe D.J. Wolfe M.S. Ann. N. Y. Acad. Sci. 2000; 920: 197-205Crossref PubMed Scopus (19) Google Scholar, 14Lanz T.A. Karmolowicz M.J. Wood K.M. Pozdnyakov N. Du P. Piotrowski M.A. Brown T.M. Nolan C.E. Richter K.E.G. Finley J.E. Fei Q. Ebbinghaus C.F. Chen Y.L. Spracklin D.K. Tate B. Geoghegan K.F. Lau L.-F. Auperin D.D. Schachter J.B. J. Pharmacol. Exp. Ther. 2006; 319: 924-933Crossref PubMed Scopus (129) Google Scholar, 15Siemers E. Skinner M. Dean R.A. Gonzales C. Satterwhite J. Farlow M. Ness D. May P.C. Clin. Neuropharmacol. 2005; 28: 126-132Crossref PubMed Scopus (230) Google Scholar). For peptide aldehyde inhibitors, some studies reported a rise that was specific for Aβ42 (3Citron M. Diehl T.S. Gordon G. Biere A.L. Seubert P. Selkoe D.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13170-13175Crossref PubMed Scopus (279) Google Scholar, 4Klafki H.-W. Abramowski D. Swoboda R. Paganetti P.A. Staufenbiel M. J. Biol. Chem. 1996; 271: 28655-28659Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 5Durkin J.T. Murthy S. Husten E.J. Trusko S.P. Savage M.J. Rotella D.P. Greenberg B.D. Siman R. J. Biol. Chem. 1999; 274: 20499-20504Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 6Zhang L. Song L. Terracina G. Liu Y. Pramanik B. Parker E. Biochemistry. 2001; 40: 5049-5055Crossref PubMed Scopus (92) Google Scholar), whereas other studies reported a rise also for Aβ40 in addition to Aβ42 (7Yamazaki T. Haass C. Saido T.C. Omura S. Ihara Y. Biochemistry. 1997; 36: 8377-8383Crossref PubMed Scopus (56) Google Scholar, 8Higaki J.N. Chakravarty S. Bryant C.M. Cowart L.R. Harden P. Scardina J.M. Mavunkel B. Luedtke J.R. Cordell B. J. Med. Chem. 1999; 42: 3889-3898Crossref PubMed Scopus (47) Google Scholar, 9Zhang L. Song L. Parker E.M. J. Biol. Chem. 1999; 274: 8966-8972Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 10Dong Y. Tan J. Cui M.-Z. Zhao G. Mao G. Singh N. Xu X. FASEB J. 2006; 20: 331-333Crossref PubMed Scopus (23) Google Scholar). However, these studies also differed as to the pharmacological target proposed to mediate the effects on Aβ; some authors considered only the protease calpain via an indirect effect on γ-secretase (7Yamazaki T. Haass C. Saido T.C. Omura S. Ihara Y. Biochemistry. 1997; 36: 8377-8383Crossref PubMed Scopus (56) Google Scholar, 9Zhang L. Song L. Parker E.M. J. Biol. Chem. 1999; 274: 8966-8972Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 10Dong Y. Tan J. Cui M.-Z. Zhao G. Mao G. Singh N. Xu X. FASEB J. 2006; 20: 331-333Crossref PubMed Scopus (23) Google Scholar), whereas others proposed a direct effect on γ-secretase (3Citron M. Diehl T.S. Gordon G. Biere A.L. Seubert P. Selkoe D.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13170-13175Crossref PubMed Scopus (279) Google Scholar, 4Klafki H.-W. Abramowski D. Swoboda R. Paganetti P.A. Staufenbiel M. J. Biol. Chem. 1996; 271: 28655-28659Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 5Durkin J.T. Murthy S. Husten E.J. Trusko S.P. Savage M.J. Rotella D.P. Greenberg B.D. Siman R. J. Biol. Chem. 1999; 274: 20499-20504Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 6Zhang L. Song L. Terracina G. Liu Y. Pramanik B. Parker E. Biochemistry. 2001; 40: 5049-5055Crossref PubMed Scopus (92) Google Scholar, 8Higaki J.N. Chakravarty S. Bryant C.M. Cowart L.R. Harden P. Scardina J.M. Mavunkel B. Luedtke J.R. Cordell B. J. Med. Chem. 1999; 42: 3889-3898Crossref PubMed Scopus (47) Google Scholar). In one study, the Aβ rise was reported in isolated membrane preparations, suggesting a direct effect of peptide aldehydes on γ-secretase (6Zhang L. Song L. Terracina G. Liu Y. Pramanik B. Parker E. Biochemistry. 2001; 40: 5049-5055Crossref PubMed Scopus (92) Google Scholar). Further evidence that γ-secretase can mediate the Aβ rise comes from studies with difluoroketone-based inhibitors, which are selective for γ-secretase and which cause a robust rise in Aβ42 both in cell culture (11Wolfe M.S. Citron M. Diehl T.S. Xia W. Donkor I.O. Selkoe D.J. J. Med. Chem. 1998; 41: 6-9Crossref PubMed Scopus (220) Google Scholar, 12Wolfe M.S. Xia W. Moore C.L. Leatherwood D.D. Ostaszewsky B.L. Rahmati T. Donkor I.O. Selkoe D.J. Biochemistry. 1999; 38: 4720-4727Crossref PubMed Scopus (309) Google Scholar, 13Moore C.L. Diehl T.S. Selkoe D.J. Wolfe M.S. Ann. N. Y. Acad. Sci. 2000; 920: 197-205Crossref PubMed Scopus (19) Google Scholar) and in isolated membrane-based assays (6Zhang L. Song L. Terracina G. Liu Y. Pramanik B. Parker E. Biochemistry. 2001; 40: 5049-5055Crossref PubMed Scopus (92) Google Scholar). Furthermore a rise in total Aβ, as well as Aβ42, in response to highly selective γ-secretase inhibitors has been observed in vivo in the plasma of guinea pigs (14Lanz T.A. Karmolowicz M.J. Wood K.M. Pozdnyakov N. Du P. Piotrowski M.A. Brown T.M. Nolan C.E. Richter K.E.G. Finley J.E. Fei Q. Ebbinghaus C.F. Chen Y.L. Spracklin D.K. Tate B. Geoghegan K.F. Lau L.-F. Auperin D.D. Schachter J.B. J. Pharmacol. Exp. Ther. 2006; 319: 924-933Crossref PubMed Scopus (129) Google Scholar) and in humans (15Siemers E. Skinner M. Dean R.A. Gonzales C. Satterwhite J. Farlow M. Ness D. May P.C. Clin. Neuropharmacol. 2005; 28: 126-132Crossref PubMed Scopus (230) Google Scholar). Thus, the biochemical mechanism of the Aβ rise has not been elucidated, and the experimental conditions required for this phenomenon have not been defined. Here we show that a rise in multiple Aβ species can be readily observed in cell cultures treated with γ-secretase inhibitors and that the key experimental requirement is a low level of βCTF or C99 expression. In addition, low substrate expression caused a shift in inhibitor potency that was independent of the Aβ rise. We also show that increased Aβ can occur in the brain following γ-secretase inhibitor dosing in rats, demonstrating the potential of γ-secretase inhibitors to cause the opposite of the intended effect in the target organ. Thus, the Aβ rise is a relevant issue in the development of γ-secretase inhibitors for Aβ-lowering therapy, and experimental conditions that exhibit the Aβ rise can be readily applied in cell culture models. Chemicals and γ-Secretase Inhibitors—Compound E (16Seiffert D. Bradley J.D. Rominger C.M. Rominger D.H. Yang F. Meredith J.E. Wang Q. Roach A.H. Thompson L.A. Spitz S.M. Higaki J.N. Prakash S.R. Combs A.P. Copeland R.A. Arneric S.P. Hartig P.R. Robertson D.W. Cordell B. Stern A.M. Olson R.E. Zaczek R. J. Biol. Chem. 2000; 275: 34086-34091Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar), DAPT (17Dovey 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. Ni B. Nissen J.S. Porter W.J. Potts B.D. Reel J.K. Stephenson D. Su Y. Shipley L.A. Whitesitt C.A. Yin T. Audia J.E. J. Neurochem. 2001; 76: 173-181Crossref PubMed Scopus (814) Google Scholar), and L-685,458 (18Li 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: 681-694Crossref PubMed Scopus (2324) Google Scholar) were purchased from Calbiochem (EMD Biosciences). BMS-299897 (19Barten D.M. Guss V.L. Corsa J.A. Loo A. Hansel S.B. Zheng M. Munoz B. Srinivasan K. Wang B. Roberts S.B. Hendrick J.P. Anderson J.J. Loy J.K. Denton R. Verdoorn T.A. Smith D.W. Felsenstein K.M. J. Pharmacol. Exp. Ther. 2005; 312: 635-643Crossref PubMed Scopus (157) Google Scholar), BMS-433796 (20Prasad C.V.C. Zheng M. Vig S. Bergstrom C. Smith D.W. Gao Q. Yeola S. Polson C.T. Corsa J.A. Guss V.L. Loo A. Wang J. Sleczka B.G. Dangler C. Robertson B.J. Hendrick J.P. Roberts S.B. Barten D.M. Bioorg. Med. Chem. Lett. 2007; 17: 4006-4011Crossref PubMed Scopus (30) Google Scholar), and DPH-068455 (21Yang M.G. Shi J.L. Modi D.P. Cochran B.M. Wolf M.A. Thompson L.A. Ramanjulu M.M. Roach A.H. Zaczek R. Robertson D.W. Wexler R.R. Olson R.E. Bioorg. Med. Chem. Lett. 2007; 17: 3910-3915Crossref PubMed Scopus (14) Google Scholar) have been described previously. DPH-111122 is a diazepinone, and BMS-267593 is an aryl sulfonamide (supplemental Fig. 1). The radioligand [3H]BMS-570479 is an aryl sulfonamide γ-secretase inhibitor that binds to the proposed allosteric inhibitor binding site (22Tian G. Ghanekar S.V. Aharony D. Shenvi A.B. Jacobs R.T. Liu X. Greenberg B.D. J. Biol. Chem. 2003; 278: 28968-28975Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 23Clarke E.E. Churcher I. Ellis S. Wrigley J.D.J. Lewis H.D. Harrison T. Shearman M.S. Beher D. J. Biol. Chem. 2006; 281: 31279-31289Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). This radioligand is dependent on presenilin for its high affinity binding in mouse embryonic fibroblast cell homogenates; binding is absent from presenilin-deficient mouse embryonic fibroblast cells (supplemental Fig. 2), which lack γ-secretase activity (24Herreman A. Serneels L. Annaert W. Collen D. Schoonjans L. De Strooper B. Nat. Cell Biol. 2000; 2: 461-462Crossref PubMed Scopus (454) Google Scholar, 25Zhang Z. Nadeau P. Song W. Donoviel D. Yuan M. Bernstein A. Yankner B.A. Nat. Cell Biol. 2000; 2: 463-464Crossref PubMed Scopus (365) Google Scholar). Cell Culture, DNA Constructs, and Transfection—HEK293 cells stably transfected with APP wild type (HEKwt) or Swedish variant (HEKsw) were derived essentially as described previously (26Selkoe D.J. Podlisny M.B. Joachim C.L. Vickers E.A. Lee G. Fritz L.C. Oltersdorf T. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7341-7345Crossref PubMed Scopus (569) Google Scholar, 27Hung A.Y. Haass C. Nitsch R.M. Qiu W.Q. Citron M. Wurtman R.J. Growdon J.H. Selkoe D.J. J. Biol. Chem. 1993; 268: 22959-22962Abstract Full Text PDF PubMed Google Scholar). Growth media and supplements were obtained from Invitrogen. For HEKwt cells, growth medium was Dulbecco's modified Eagle's medium (DMEM) containing 10% heat inactivated fetal bovine serum, 2 mm l-glutamine, 1 mm sodium pyruvate, 400 μg/ml G418, and 5 μg/ml blasticidin. Growth medium for HEKsw cells was the same as for HEKwt cells but containing 200 μg/ml hygromycin B and lacking blasticidin. THP-1 cells were grown in roller bottles in RPMI 1640 medium containing l-glutamine and 10 μm β-mercaptoethanol to a density of 1 × 106/ml. Cells were harvested by centrifugation, and cell pellets were quick frozen in dry ice/ethanol and stored at –80 °C prior to use. Mouse embryonic fibroblasts were passaged twice per week in a 1:1 mixture of DMEM and F-12 nutrient mixture supplemented with 10% fetal bovine serum, penicillin, and streptomycin. HeLa cells were maintained in DMEM containing 10% fetal bovine serum, penicillin, streptomycin, and 2 mm l-glutamine. For inhibitor treatments, cell cultures were grown for 24 h, and the medium was replaced with DMEM containing high glucose, 0.0125% bovine serum albumin, nonessential amino acids, 2 mm l-glutamine, and 100 units/ml penicillin and streptomycin. Inhibitors were added in DMSO to a final concentration of 0.1% DMSO. Treated cells were incubated overnight at 37 °C in a humidified atmosphere containing 5% CO2. Inhibitor treatments were carried out in 96-well format for Figs. 1, 3, and 4, in cell culture flasks for Fig. 2, and in 24-well format for Fig. 6. For transient expression of C99, the βCTF sequence (99 C-terminal residues of APP) was preceded by a signal peptide containing the amino acid sequence MLPGLALLLLAAYTARADA and followed by the c-Myc epitope tag, EQKLISEEDL, at the C-terminal end. The DNA construct encoding this sequence was cloned into the expression vector pCDNA3.1 (Invitrogen). HeLa cell cultures were transiently transfected in T 162-cm2 flasks with the C99 expression construct using TransIT-HelaMONSTER (Mirus) according to the manufacturer's directions. A total of 36 μg of DNA was used in each transfection consisting of a mixture of vector DNA and either 0.9, 1.8, or 36 μg of the C99 expression construct DNA. After incubation overnight, transfected cells were detached using trypsin/EDTA for 2 min at ambient temperature, collected by centrifugation, and resuspended in growth medium. Cells were again collected by centrifugation; resuspended in defined medium consisting of DMEM, 2 mm l-glutamine, penicillin, streptomycin, 0.0125% bovine serum albumen, and nonessential amino acids; and then replated in 96-well plates at 200 μl/well (Packard View Plate, 96-well, black).FIGURE 3The C99 expression level affects the Aβ rise and inhibitory potency of γ-secretase inhibitors. A, HeLa cell cultures were transfected with different amounts of the C99 DNA construct, and Aβ-(1–40) secreted into the medium was quantified. Error bars, all smaller than symbols used, represent S.D. for 19 independent experiments. Linear regression indicated a goodness of fit, r2, equal to 0.95. B, HEKwt cell cultures were transfected with 1.8 μg(•) or 36 μg(▴) of C99 DNA construct and then treated with DAPT at a range of concentrations for 16 h. The level of Aβ-(1–40) secreted into the culture medium is expressed as a percentage of Aβ-(1–40) in vehicle-treated control cultures. Error bars represent S.D. for five independent experiments. C, HEKwt cell cultures were transfected with 0.9 μg(▾), 1.8 μg(•), or 36 μg (▴) of C99 DNA construct and then treated with γ-secretase inhibitor BMS-267593 at a range of concentrations for 16 h. The level of Aβ-(1–40) secreted into the culture medium is expressed as a percentage of Aβ-(1–40) in DMSO-treated control cultures. Error bars represent S.D. for four independent experiments. D, same experimental design as C except that the cell cultures were treated with γ-secretase inhibitor compound E (CPDE), and error bars represent S.D. for five independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Inhibitors of γ-secretase in cell-free assays. A, γ-secretase cell-free assays were conducted in the presence of DAPT at an enzyme to substrate ratio of 1:1 (•) or 1:100 (▴). Error bars indicate S.D. for three independent experiments. The IC50 values for DAPT were 7.9 ± 0.6 and 8.6 ± 0.9 nm at enzyme to substrate ratios of 1:1 and 1:100, respectively. B, same as A except that L-685,458 was the inhibitor used. The IC50 values for L-685,458 were 4.3 ± 0.6 and 0.1 ± 0.02 nm at enzyme to substrate ratios of 1:1 and 1:100, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 2LC/MS analysis of Aβ peptides secreted by HEKsw and HEKwt cell cultures and the Aβ rise in HEKwt cells. A, Aβ peptide species in HEKsw cell medium were quantified by LC/MS. The intensity of each peptide is expressed relative to the [15N]Aβ-(1–40) internal control. Error bars represent the S.D. for three measurements. B, Aβ peptide species in HEKwt cell medium were quantified by LC/MS as described in A. Panels C, D, E, and F, HEKwt cells were treated with a range of concentrations of DAPT, and Aβ species were quantified by LC/MS. Percentages are expressed relative to the quantity of each peptide in vehicle-treated control cultures except for Aβ-(1–19) for which the percentage is relative to the amount detected in the presence of 10 nm DAPT. C, Aβ-(1–37) (▴) and Aβ-(17–37) (•). D, Aβ-(1–38) (▴) and Aβ-(17–38) (•). E, Aβ-(1–40) (▴), Aβ-(17–40) (•), and Aβ-(11–40) (▾). F, Aβ-(1–19) (▴) and Aβ-(17–28) (•). Note that levels of Aβ-(1–42) were below the level of quantitation by this method.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6Quantitation of γ-secretase enzyme and βCTF concentrations. A, saturation binding isotherms for [3H]BMS-570479 were determined in homogenates from HEKsw (○) and HEKwt (▵) cultures. Error bars represent S.E. for three independent experiments. The dissociation binding constant, Kd, was 0.6 ± 0.15 and 0.6 ± 0.2 nm in the HEKsw and HEKwt homogenates, respectively. The maximal number of binding sites, Bmax, was 410 ± 45 and 370 ± 70 fmol/mg of total protein in the HEKsw and HEKwt homogenates, respectively. B, the graph shows the linear response of recombinant C100 determined by Western blotting. The concentration of βCTF in HEKwt and HEKsw cells was determined by Western blotting from this linear calibration. C, the graph shows the linear response of synthetic Aβ-(17–28) peptide determined by LC/MS. The concentration of Aβ-(17–28) peptide derived from recombinant C100 digestion by trypsin was determined by LC/MS from this linear calibration.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cell-free γ-Secretase Assay—Assays were carried out based on a procedure described previously using C99 substrate expressed and purified from Escherichia coli (28Tian G. Sobotka-Briner C.D. Zysk J. Liu X. Birr C. Sylvester M.A. Edwards P.D. Scott C.D. Greenberg B.D. J. Biol. Chem. 2002; 277: 31499-31505Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). To prepare concentrated γ-secretase enzyme, lipid rafts were isolated based on methods described previously (29Wahrle S. Das P. Nyborg A.C. McLendon C. Shoji M. Kawarabayashi T. Younkin L.H. Younkin S.G. Golde T.E. Neurobiol. Dis. 2002; 9: 11-23Crossref PubMed Scopus (370) Google Scholar). Briefly THP-1 cell pellets were lysed in 4 volumes of lysis buffer (125 mm NaCl, 1% CHAPSO, 25 mm Na-MES, pH 6.5) containing a protease inhibitor mixture of 104 μm 4-(2-aminoethyl)benzenesulfonyl fluoride, 80 nm aprotinin, 2 μm leupeptin, 4 μm bestatin, 1.5 μm pepstatin A, and 1.4 μm E-64 (0.1% protease inhibitor mixture P8340, Sigma-Aldrich) using five passages through a 25-gauge needle, and the lipid raft fraction was isolated by discontinuous sucrose density gradient centrifugation (29Wahrle S. Das P. Nyborg A.C. McLendon C. Shoji M. Kawarabayashi T. Younkin L.H. Younkin S.G. Golde T.E. Neurobiol. Dis. 2002; 9: 11-23Crossref PubMed Scopus (370) Google Scholar). This procedure yielded a stock preparation of CHAPSO-solubilized proteins containing γ-secretase at a concentration of 3 nm as determined by saturation radioligand binding. γ-Secretase activity assays were performed in assay buffer (100 nm NaCl, 0.25% CHAPSO, 50 mm HEPES, pH 7.0). The lipid raft preparation was mixed with C99 substrate at molar ratios of 1:1 and 1:100, corresponding to absolute concentrations of 0.3 nm:0.3 nm and 0.01 nm:1 nm, respectively, at a volume of 200 μl/reaction in 96-well polypropylene plates. After incubation for 3 h at 37 °C, Aβ-(1–40) was quantified by enzyme-linked immunosorbent assay (ELISA). Extensive experimentation to optimize assay conditions showed that the maximum concentration of γ-secretase that could be used in the assay was 0.3 nm because of inhibition of Aβ production at higher concentrations presumably due to inhibitory activities present in the raft preparation. Likewise C99 substrate at concentrations less than 0.03 nm yielded insufficient signal for reliable quantification of Aβ-(1–40). Thus, the highest enzyme to substrate ratio that could be utilized using this method was 1:1. Aβ-(1–40) was quantified by ELISA using the antibodies TSD9S3.2 and 26D6 described below. Radioligand Binding in Cell Homogenates—HEKsw, HEKwt, and mouse embryonic fibroblast cell pellets were homogenized in 10 ml of 50 mm HEPES with 0.1% protease inhibitor mixture (Sigma-Aldrich P8340) at pH 7.0 and 4 °C using a Dounce homogenizer. The homogenate was centrifuged at 48,000 × g for 20 min. Protein determinations were carried out using a Bradford based assay (Bio-Rad). The final pellet was resuspended in buffer to yield a protein concentration of 5 mg/ml. [3H]BMS-570479 binding was carried out in 50 mm HEPES, 0.1% CHAPSO, pH 7.0, at a protein concentration of 200 μg/ml. Binding assays were performed in polypropylene 96-deepwell plates (Beckman Coulter, Fullerton, CA) in a final volume of 0.25 ml containing 5% (v/v) DMSO. Assays were initiated by the addition of 25 μl of assay buffer containing radioligand to 12.5 μl of dimethyl sulfoxide containing various concentrations of unlabeled compounds followed by 212 μl of cell homogenate. Nonspecific binding was defined in the presence of 1 μm BMS-433796. After incubating at 25 °C for 1.5 h, bound radioligand was separated from free by filtration over GF/B glass fiber filters (Brandel, Gaithersburg, MD) presoaked in phosphate-buffered saline (PBS), pH 7.0, using a cell harvester (Brandel). Filters were washed four times with 1.0 ml of ice-cold PBS,
HIV-1 maturation inhibitors (MIs) disrupt the final step in the HIV-1 protease-mediated cleavage of the Gag polyprotein between capsid p24 capsid (CA) and spacer peptide 1 (SP1), leading to the production of infectious virus. BMS-955176 is a second generation MI with improved antiviral activity toward polymorphic Gag variants compared to a first generation MI bevirimat (BVM). The underlying mechanistic reasons for the differences in polymorphic coverage were studied using antiviral assays, an LC/MS assay that quantitatively characterizes CA/SP1 cleavage kinetics of virus like particles (VLPs) and a radiolabel binding assay to determine VLP/MI affinities and dissociation kinetics. Antiviral assay data indicates that BVM does not achieve 100% inhibition of certain polymorphs, even at saturating concentrations. This results in the breakthrough of infectious virus (partial antagonism) regardless of BVM concentration. Reduced maximal percent inhibition (MPI) values for BVM correlated with elevated EC50 values, while rates of HIV-1 protease cleavage at CA/SP1 correlated inversely with the ability of BVM to inhibit HIV-1 Gag polymorphic viruses: genotypes with more rapid CA/SP1 cleavage kinetics were less sensitive to BVM. In vitro inhibition of wild type VLP CA/SP1 cleavage by BVM was not maintained at longer cleavage times. BMS-955176 exhibited greatly improved MPI against polymorphic Gag viruses, binds to Gag polymorphs with higher affinity/longer dissociation half-lives and exhibits greater time-independent inhibition of CA/SP1 cleavage compared to BVM. Virological (MPI) and biochemical (CA/SP1 cleavage rates, MI-specific Gag affinities) data were used to create an integrated semi-quantitative model that quantifies CA/SP1 cleavage rates as a function of both MI and Gag polymorph. The model outputs are in accord with in vitro antiviral observations and correlate with observed in vivo MI efficacies. Overall, these findings may be useful to further understand antiviral profiles and clinical responses of MIs at a basic level, potentially facilitating further improvements to MI potency and coverage.
Stable isotope labeling of proteins affords indicators at the molecular level, specifically biomarkers, which may providein vivodata on disease diagnosis, progression, and treatment.
The LC-MS bioanalysis of protein kinetics assays is simplified by a data normalization strategy via internal proteolytic analyte utilized as a control standard.
Plasma pyridoxic acid (PDA) and homovanillic acid (HVA) were recently identified as novel endogenous biomarkers of organic anion transporter (OAT) 1/3 function in monkeys. Consequently, this clinical study assessed the dynamic changes and utility of plasma PDA and HVA as an initial evaluation of OAT1/3 inhibition in early-phase drug development. The study was designed as a single-dose randomized, three-phase, crossover study; 14 Indian healthy volunteers received probenecid (PROB) (1000 mg orally) alone, furosemide (FSM) (40 mg orally) alone, or FSM 1 hour after receiving PROB (40 and 1000 mg orally) on days 1, 8, and 15, respectively. PDA and HVA plasma concentrations remained stable over time in the prestudy and FSM groups. Administration of PROB significantly increased the area under the plasma concentration-time curve (AUC) of PDA by 3.1-fold (dosed alone; P < 0.05), and 3.2-fold (coadministered with FSM; P < 0.01), compared with the prestudy and FSM groups, respectively. The corresponding increase in HVA AUC was 1.8-fold (P > 0.05) and 2.1-fold (P < 0.05), respectively. The increases in PDA AUC are similar to those in FSM AUC, whereas those of HVA are smaller (3.1–3.2 and 1.8–2.1 vs. 3.3, respectively). PDA and HVA renal clearance (CLR) values were decreased by PROB to smaller extents compared with FSM (0.35–0.37 and 0.67–0.73 vs. 0.23, respectively). These data demonstrate that plasma PDA is a promising endogenous biomarker for OAT1/3 function and that its plasma exposure responds in a similar fashion to FSM upon OAT1/3 inhibition by PROB. The magnitude and variability of response in PDA AUC and CLR values between subjects is more favorable relative to HVA.
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