Abstract
Selective lowering of Aβ42 levels (the 42-residue isoform of the amyloid-β peptide) with small-molecule γ-secretase modulators (GSMs), such as some non-steroidal anti-inflammatory drugs, is a promising therapeutic approach for Alzheimer’s disease1. To identify the target of these agents we developed biotinylated photoactivatable GSMs. GSM photoprobes did not label the core proteins of the γ-secretase complex, but instead labelled the β-amyloid precursor protein (APP), APP carboxy-terminal fragments and amyloid-β peptide in human neuroglioma H4 cells. Substrate labelling was competed by other GSMs, and labelling of an APP γ-secretase substrate was more efficient than a Notch substrate. GSM interaction was localized to residues 28–36 of amyloid-β, a region critical for aggregation. We also demonstrate that compounds known to interact with this region of amyloid-β act as GSMs, and some GSMs alter the production of cell-derived amyloid-β oligomers. Furthermore, mutation of the GSM binding site in the APP alters the sensitivity of the substrate to GSMs. These findings indicate that substrate targeting by GSMs mechanistically links two therapeutic actions: alteration in Aβ42 production and inhibition of amyloid-β aggregation, which may synergistically reduce amyloid-β deposition in Alzheimer’s disease. These data also demonstrate the existence and feasibility of ‘substrate targeting’ by small-molecule effectors of proteolytic enzymes, which if generally applicable may significantly broaden the current notion of ‘druggable’ targets2.
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Acknowledgements
This work was supported by grants from the US National Institutes of Health National Institute on Aging (to D.M.W., M.S.W., T.E.G., A.F. and E.H.K.), the AFAR CART award (T.E.G.), and the Mayo Foundation. T.L.K. was supported by the American Health Assistance Foundation ADR program, the Mayo ADRC, and a Robert and Clarice Smith Fellowship. A.T.W. was funded by a European Union 6th Framework Marie Curie Early Stage Training fellowship. P.C.F was supported by the Swiss National Science Foundation and by the NCCR ‘Neural Plasticity and Repair’. 21F12 was a gift from P. Seubert and D. Schenk.
Author Contributions T.L.K., D.M.W., E.H.K. and T.E.G. conceived the project. T.L.K., T.B.L. and M.A.B. planned, performed and analysed the photoaffinity experiments and amyloid-β assays. P.C.F., W.Y., and M.S.W. performed photoaffinity experiments with purified γ-secretase and APP(C100)–Flag and Notch(C100)–Flag; T.L.K. wrote most of the paper. T.L.K., M.A.B., C.V. and R.W.P. performed and analysed mouse experiments. K.J.-W. cloned constructs and made stable cell lines. S.S., B.C., J.T. and E.H.K. made the APP-NOTCH chimaera construct and cell line and performed mass spectrometry analysis of amyloid-β. A.T.W. and D.M.W. analysed amyloid-β oligomers. D.Y. and C.E. screened and provided compound libraries. G.M.M., B.H., R.C., and A.F. synthesized fenofibrate and various photoprobes. J.E. characterized initial photoprobes. R.N. and B.S. synthesized flurbiprofen and related photoprobes. B.M., V.R., B.C. and T.L.R. performed mass spectrometry analysis of amyloid-β, APP-NOTCH amyloid-β and saturation binding experiments with X-34.
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E.H.K. and T.E.G. are co-inventors on a patent licensed to Myriad Genetics with claims pertaining to the use of Aβ42-lowering NSAIDs and NSAID derivatives in Alzheimer’s disease.
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The file contains Supplementary Figures 1-13 with Legends, a Supplementary Discussion, and Supplementary Methods describing the detailed synthesis and characterization of chemicals used in the study. (PDF 2089 kb)
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Kukar, T., Ladd, T., Bann, M. et al. Substrate-targeting γ-secretase modulators. Nature 453, 925–929 (2008). https://doi.org/10.1038/nature07055
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DOI: https://doi.org/10.1038/nature07055
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