Sulindac Sulfide Is a Noncompetitive γ-Secretase Inhibitor That Preferentially Reduces Aβ42 Generation

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been known to reduce risk for Alzheimer's disease. In addition to the anti-inflammatory effects of NSAIDs to block cylooxygenase, it has been shown recently that a subset of NSAIDs selectively inhibits the secretion of highly amyloidogenic Aβ42 from cultured cells, although the molecular target(s) of NSAIDs in reducing the activity of γ-secretase for Aβ42 generation (γ42-secretase) still remain unknown. Here we show that sulindac sulfide (SSide) directly acts on γ-secretase and preferentially inhibits the γ42-secretase activity derived from the 3-(3-cholamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate-solubilized membrane fractions of HeLa cells, in an in vitroγ-secretase assay using recombinant amyloid β precursor protein C100 as a substrate. SSide also inhibits activities for the generation of Aβ40 as well as for Notch intracellular domain at higher concentrations. Notably, SSide displayed linear noncompetitive inhibition profiles for γ42-secretase in vitro. Our data suggest that SSide is a direct inhibitor of γ-secretase that preferentially affects the γ42-secretase activity. Nonsteroidal anti-inflammatory drugs (NSAIDs) have been known to reduce risk for Alzheimer's disease. In addition to the anti-inflammatory effects of NSAIDs to block cylooxygenase, it has been shown recently that a subset of NSAIDs selectively inhibits the secretion of highly amyloidogenic Aβ42 from cultured cells, although the molecular target(s) of NSAIDs in reducing the activity of γ-secretase for Aβ42 generation (γ42-secretase) still remain unknown. Here we show that sulindac sulfide (SSide) directly acts on γ-secretase and preferentially inhibits the γ42-secretase activity derived from the 3-(3-cholamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate-solubilized membrane fractions of HeLa cells, in an in vitroγ-secretase assay using recombinant amyloid β precursor protein C100 as a substrate. SSide also inhibits activities for the generation of Aβ40 as well as for Notch intracellular domain at higher concentrations. Notably, SSide displayed linear noncompetitive inhibition profiles for γ42-secretase in vitro. Our data suggest that SSide is a direct inhibitor of γ-secretase that preferentially affects the γ42-secretase activity. Alzheimer's disease amyloid β peptide amyloid-β precursor protein 3-(3-cholamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate cyclooxygenase N-N-(3,5-difluorophenacetyl)-l-alanyl-(S)-phenylglycinet-butyl ester enzyme-linked immunosorbent assay Notch intracellular domain nonsteroidal anti-inflammatory drugs mutant presenilin sulindac sulfide sulindac sulfone wild-type N,N-bis(2-hydroxyethyl)glycine high molecular weight APP intracellular domain peroxisome proliferator-activated receptors Alzheimer's disease (AD)1 is a dementing neurodegenerative disorder of the elderly characterized pathologically by neuronal loss in the cerebral cortex accompanied by massive deposition of amyloid β peptides (Aβ) as senile plaques (1Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5196) Google Scholar). Aβ is produced by sequential proteolytic cleavages of the amyloid β precursor protein (βAPP) by a set of membrane-bound proteases termed β- and γ-secretases. The C-terminal length of Aβ generated by γ-secretase is heterogeneous; Aβ42 is a relatively minor molecular species of the Aβ secreted from cells, but it has a much higher propensity to aggregate and form amyloid compared with other Aβ species. These findings provide strong support for the hypothesis that the deposition of Aβ42 is closely related to the pathogenesis of AD, implicating γ-secretase as an important therapeutic target. Mutations in PS1 or PS2 genes account for the majority of early onset familial AD, and these mutations cause an increase in the ratio or levels of production of Aβ42 (1Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5196) Google Scholar). It is known that PS is essential for the γ-secretase-mediated intramembranous cleavage not only for βAPP but for other type I transmembrane proteins (e.g. Notch, ErbB4, E-cadherin, low density lipoprotein receptor-related protein, and CD44) (2Fortini M.E. Nat. Rev. Mol. Cell. Biol. 2002; 3: 673-684Crossref PubMed Scopus (345) Google Scholar). PS proteins undergo endoproteolysis to generate N- and C-terminal fragments and interact with other proteins (i.e. nicastrin, APH-1, and PEN-2) to form a high molecular weight (HMW) protein complex (3Haass C. Steiner H. Trends Cell Biol. 2002; 12: 556-562Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The functional role of PS complex in γ-secretase activity still remains unknown. However, aspartyl protease transition state analogue inhibitors of γ-secretase, which harbors a hydroxyl ethylene isostere or a difluoro alcohol moiety, directly label PS fragments (4Li 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 (867) Google Scholar, 5Esler W.P. Kimberly W.T. Ostaszewski B.L. Diehl T.S. Moore C.L. 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Notably, a proteolytic generation of Notch intracellular domain by γ-secretase is the well known and effects by the of Notch by γ-secretase inhibitors are N.R. D. Moore C.L. Wolfe M.S. Kopan R. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar, P. Shearman M.S. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). the γ-secretase activity to generate in we generated a recombinant of a of to the cleavage and transmembrane domain to a C-terminal of with the membrane we the of an which by treatment with in a and data not the recombinant is also by γ-secretase to generate in which with the by Wolfe and W.P. Kimberly W.T. C. A. N. Wolfe M.S. J. PubMed Scopus Google Scholar, W.T. W.P. Ye W. Ostaszewski B.L. J. Diehl T. Selkoe D.J. Wolfe M.S. PubMed Scopus Google Scholar). It has been recently S. J.L. P. Wang R. Smith T. N. Nature. 2001; PubMed Scopus Google Scholar, T. T. W. Cole G.M. J. 2002; PubMed Scopus Google that a subset of NSAIDs Aβ42 Notch in cultured the of NSAIDs on APP and Notch we have sulindac sulfide sulindac sulfone and to and T. R. Takikawa R. Iwatsubo T. 2002; PubMed Scopus Google Scholar). a in Aβ42 secretion by treatment with the secretion of Aβ40 as well as Notch not with SSide by in in Aβ generation as well as in protein I. J. G. J. 2001; PubMed Scopus Google Scholar). The for Aβ42 secretion of SSide and on Aβ40 or Aβ42 secretion as well as on Notch cleavage to SSide γ-secretase activity by direct or (e.g. the of or the secretion or of Aβ42 or the of we the in vitro γ-secretase activity in membrane in the of an inhibition of γ42-secretase activity by SSide in a The of SSide for γ42-secretase activity in a in by the increase of the of SSide in the of the that SSide is not an or inhibitor we the γ-secretase with SSide γ-secretase activity these it that the molecular of SSide is the γ-secretase complex and that SSide as a γ-secretase In to the in cultured cells, the of SSide at low a but increase in Aβ40 generation in vitro SSide the of in addition to that of at high SSide has an inhibition activity at high it the activity at It that the in Aβ42 secretion by SSide accompanied by a increase in secretion S. J.L. P. Wang R. Smith T. N. Nature. 2001; PubMed Scopus Google Scholar). the in Aβ42 production by high of SSide affects that of in we the generated Aβ species by high J. A. B. G. U. M. G. J. PubMed Scopus Google Scholar). a increase in generation in the low accompanied by a in Aβ42 although the in vitro generation of Aβ peptides by high of SSide not These suggest that SSide is a γ-secretase inhibitor directly the membrane-embedded protease and activities of the of γ42-secretase activity by we the analysis that at a but of that SSide displayed a noncompetitive inhibition for γ42-secretase activity. transition state analogue γ-secretase inhibitors (i.e. or displayed linear noncompetitive inhibition a for intramembranous cleavage by γ-secretase has been G. J. C. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). In γ-secretase complex is to harbor a of as well as a catalytic SSide affects γ42-secretase activity by with the catalytic of we the inhibition of SSide by with a transition state analogue inhibitor that is to the of SSide to the with SSide and in a inhibition of γ42-secretase and cause an increase in the inhibition an addition of on the the that SSide the γ42-secretase activity in a to that of a transition state analogue SSide for a (i.e. catalytic with in the γ42-secretase complex the of of the inhibitors affects the of the the of SSide on intracellular domain generation from recombinant of βAPP and Notch in that the inhibition of generation from by SSide to that of the Aβ the proteolytic activity to from by treatment with it at In production from by SSide in a at the of SSide on generation from recombinant in vitro. The cleavage of by high of SSide it not by SSide at to which with the in cultured These that SSide the γ-secretase activity for Notch as well as for although inhibition for Notch is much that for for It has been shown that a subset of NSAIDs selectively the secretion of although the molecular NSAIDs the γ-secretase activity S. J.L. P. Wang R. Smith T. N. Nature. 2001; PubMed Scopus Google Scholar, T. T. W. Cole G.M. J. 2002; PubMed Scopus Google Scholar). In we an in vitro γ-secretase assay using recombinant type as well as mutant C100 as and the of that but not directly inhibits the γ-secretase activity derived from membrane fractions of HeLa in a we that SSide is a inhibitor that has the inhibition activity compared with for or Notch, with noncompetitive inhibition It has been that NSAIDs molecular of which the is that to I. J. G. J. 2001; PubMed Scopus Google Scholar). In addition, SSide has been shown to β and reduce the and of I. J. G. J. 2001; PubMed Scopus Google Scholar). It has been shown that the of NSAIDs is of activity in cultured S. J.L. P. Wang R. Smith T. N. Nature. 2001; PubMed Scopus Google Scholar). the inhibition of Aβ secretion in by although the analysis of the inhibition at high of the SSide directly inhibits γ-secretase, we an in vitro assay using as and recombinant C100 as and we that SSide has the to the γ-secretase although cleavage at Aβ42 In addition, in vitro Aβ generation in the of that activities (e.g. are not in the of γ-secretase by we also that addition of not the of Aβ that γ-secretase activity not Y. and T. Finally, we that SSide displayed noncompetitive inhibition which is a of a of γ-secretase inhibitors G. J. C. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). these we that SSide as a γ-secretase inhibitor that directly affects activity by to the membrane-embedded protease γ-secretase inhibitors have been to secretion of Aβ40 and Aβ42 in The peptide and inhibitors a or alcohol increase Aβ42 secretion at and it at a high Aβ40 generation in an M.S. M. Diehl T.S. Xia W. Selkoe D.J. J. Chem. 1998; PubMed Scopus Google Scholar, S. S.P. R. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, L. L. G. Y. B. 2001; PubMed Scopus Google Scholar). However, the of of peptide Aβ40 and Aβ42 are at that a γ-secretase complex generate Aβ40 and Aβ42 S. S.P. R. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In addition, the transition state analogue inhibitors of aspartyl proteases a isostere also the secretion of Aβ40 and Aβ42 to (12Li 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 (501) Google Scholar, L. L. G. Y. B. 2001; PubMed Scopus Google Scholar). The inhibition a of the membrane that harbors and and an in vitro assay using recombinant that these directly on γ-secretase and that the in the inhibition on or within the γ-secretase The molecular the in Aβ40 and Aβ42 generation at low of peptide inhibitors and SSide still remains unknown. is that a loss of by the low of peptide or peptide the to γ42-secretase that is still to of In SSide inhibition profiles to preferentially Aβ42 which accompanied by an increase in the production of as well as Aβ40 at in vitro. that SSide on a that is from by peptide or protease SSide and other inhibitors effects on a that is in the of the of a to in γ-secretase a study with the transition state analogue a direct with the that SSide directly on the catalytic although the of SSide is not to known that SSide to the of γ-secretase complex and the catalytic in a to and an direct of proteolytic activities by protease inhibitors or has been in that harbors proteolytic activities and A.L. Mol. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, G. S. M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, J. K. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). an inhibitor of protease, inhibits the activity is G. S. M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). studies using inhibitors that (e.g. that cause on to an studies using of SSide that are to that SSide acts directly on It has been that γ-secretase inhibitors the cleavage of Notch in cultured cells, with the of SSide and a S. J.L. P. Wang R. Smith T. N. Nature. 2001; PubMed Scopus Google A. F. F. J.L. Nat. Cell Biol. 2001; 3: PubMed Scopus Google Scholar). However, it has been shown that to γ-secretase activity in that is not a direct inhibitor of γ-secretase W.P. C. Kimberly W.T. Diehl T.S. Ye W. Ostaszewski B.L. Xia W. Selkoe D.J. Wolfe M.S. Nat. Cell Biol. 2002; PubMed Scopus Google Scholar). that SSide has the to the endoproteolysis of Notch at the at much higher compared with for Aβ inhibition in other inhibitors (e.g. Notch cleavage with to for Aβ These suggest that and Notch are but it to the effects of γ-secretase inhibitors by inhibition of Notch by of to the molecular of inhibition on γ-secretase activity by SSide and to to βAPP cleavage not only the development of a therapeutic for AD but also of the intramembranous proteolytic activity of γ-secretase that proteins at Y. B. De and R. Kopan for and and for support for