Abstract
γ-Secretase, exhibiting characteristics of aspartyl protease, mediates the intramembranous proteolysis of β-amyloid precursor protein (APP) and Notch, and it is considered to be a prime pharmacological target in the development of therapeutics for Alzheimer's disease (AD). To identify compounds that block γ-secretase-mediated proteolysis, we used a highly sensitive cell-based reporter gene assay for γ-secretase in which Gal4/VP16-tagged C99-APP was expressed as the immediate substrate of γ-secretase, and Gal4/VP16-tagged APP intracellular domain released by the γ-secretase cleavage then activated the expression of the Gal4-driven luciferase reporter gene. Using this reporter assay, we demonstrated that the newly synthesized (hydroxyethyl)urea peptidomimetics, which contain unnatural amino acid moieties at positions P1′ and/or P3′, can effectively inhibit γ-secretase activity and significantly reduce Aβ production. The γ-secretase-dependent S3 cleavage of Notch was also consistently blocked by these (hydroxyethyl)ureas as evidenced by the decreased generation of the Notch intracellular domain, a prerequisite for the activation of Notch signaling. The inhibition of Notch signaling by active Jia compounds efficiently promotes the neuronal differentiation of neuroblastoma cells, intervening in tumorigenesis and the malignancy of neuroblastomas. Our results suggest that (hydroxyethyl)urea peptidomimetics containing unnatural amino acid substitutions could represent a novel class of γ-secretase inhibitors with enhanced stability, providing the basis for the further development of effective therapeutics for AD and neuroblastomas.
Footnotes
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This study was supported by National Science Council, Taiwan, grants NSC 93-2320-B-001-037, NSC 94-2320-B-001-004, NSC 94-3112-B-001-001, and NSC 95-3112-B-001-006 (to Y.-F.L.) and NSC 92-2320-B-016-046 (to M.-K.H.) and Academia Sinica (to Y.-F.L.). Proteomic mass spectrometry analyses performed by the Core Facilities for Proteomics Research located at the Institute of Biological Chemistry, Academia Sinica, were supported by National Science Council Grant NSC 91-3112-P-001-009-Y and the Academia Sinica.
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ABBREVIATIONS: AD, Alzheimer's disease; PS, presenilin; NTF, N-terminal fragment; CTF, C-terminal fragment; NICD, Notch intracellular domain; HIV, human immunodeficiency virus; ELISA, enzyme-linked immunosorbent assay; HEK, human embryonic kidney; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; DMSO, dimethyl sulfoxide; APP, amyloid precursor protein; C99, 99-residue C-terminal fragment of amyloid precursor protein; PBS, phosphate-buffered saline; PLB, passive lysis buffer; DAPT, N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester; GV, Gal4/VP16 DNA binding domain; GAP-43, growth-associated protein 43; PAGE, polyacrylamide gel electrophoresis; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBST, phosphate-buffer saline/Tween 20; Aβ, β-amyloid; HRP, horseradish peroxidase; CHAPSO, 3-[(3-cholamidopropyl)dimethyl-ammonio]-2-hydroxy-1-propanesulfonate; siRNA, small interfering RNA; RNAi, RNA interference; ESI, electrospray ionization; MS, mass spectrometry, MS/MS, tandem mass spectrometry; LC, liquid chromatography; OD, optical density; Chy, cyclohexylmethyl; RA, retinoic acid; L-685-458, (2R-(2R*,4R*,5S*))N-(5-(((1,1-dimethylethoxy)carbonyl)amino)-4-hydroxy-1-oxo-6-phenyl-2-(phenylmethyl)hexyl)-l-leucyl-l-phenylalaninamide.
- Received March 9, 2006.
- Accepted November 14, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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Unnatural Amino Acid-Substituted (Hydroxyethyl)urea Peptidomimetics Inhibit γ-Secretase and Promote the Neuronal Differentiation of Neuroblastoma Cells
