First and second generation γ-secretase modulators (GSMs) modulate amyloid-β (Aβ) peptide production through different mechanisms

Abstract

γ-Secretase-mediated cleavage of amyloid precursor protein (APP) results in the production of Alzheimer disease-related amyloid-β (Aβ) peptides. The Aβ42 peptide in particular plays a pivotal role in Alzheimer disease pathogenesis and represents a major drug target. Several γ-secretase modulators (GSMs), such as the nonsteroidal anti-inflammatory drugs (R)-flurbiprofen and sulindac sulfide, have been suggested to modulate the Alzheimer-related Aβ production by targeting the APP. Here, we describe novel GSMs that are selective for Aβ modulation and do not impair processing of Notch, EphB2, or EphA4. The GSMs modulate Aβ both in cell and cell-free systems as well as lower amyloidogenic Aβ42 levels in the mouse brain. Both radioligand binding and cellular cross-competition experiments reveal a competitive relationship between the AstraZeneca (AZ) GSMs and the established second generation GSM, E2012, but a noncompetitive interaction between AZ GSMs and the first generation GSMs (R)-flurbiprofen and sulindac sulfide. The binding of a (3)H-labeled AZ GSM analog does not co-localize with APP but overlaps anatomically with a γ-secretase targeting inhibitor in rodent brains. Combined, these data provide compelling evidence of a growing class of in vivo active GSMs, which are selective for Aβ modulation and have a different mechanism of action compared with the original class of GSMs described.

FIGURE 1.

Chemical structures of compounds explored in this study. A, AZ4800; B, AZ3303; C, Sulindac Sulfide; D, AZ1136; E, E-2012; F, (R)-flurbiprofen; G, [³H]AZ8349; and H, [³H]DBZ.

FIGURE 2.

Effect of second generation GSMs on APP processing in cellular and cell-free assays. A, Aβ40 and Aβ42 measurement in conditioned media from HEK/APPswe cells. All AZ GSMs and GSM E2012 inhibit Aβ42 (black) and Aβ40 (gray) generation. B–F, Aβ measurement in conditioned media from HEK/APPswe cells. B, AZ1136 increases the levels of Aβ37 and Aβ39. C, AZ4800 increases Aβ37 > Aβ38 and lowers Aβ39. D, AZ3303 increases Aβ38 > Aβ37 and lowers Aβ39. E, E2012 increases Aβ37. AZ1136 (F) and AZ4800 (G) display Aβ modulation in cell membranes derived from HEK/APPswe cells. % Aβ release is set relative to 0.5% DMSO (100%) and 0.5 μm L685458 (0%) controls (mean ± S.E., n = 2).

FIGURE 3.

Effect of second generation GSMs on Notch, EphB2, and EphA4 processing. A, none of the AZ GSMs (square, triangle, and ×) or E2012 (○) affect Notch processing, whereas the GSIs L685458, DBZ, MRK-560, and Semagacestat do, as measured by quantifying the amount of nuclearly translocated NICD in HEK293 cells stably transfected with ΔENotch using immunocytochemistry. % NICD translocation is set relative to 0.5% DMSO (100%) and 0.5 μm L685458 (0%) controls (mean ± S.E., n = 2). B, HEK293 cells, transiently transfected with Myc-tagged EphB2, EphA4, and ΔENotch constructs, were exposed to 100 nm DBZ or 1 μm 4800 for 15 h prior to Western blot analysis. DBZ, but not AZ4800, results in accumulation of respective protein, as explored with an anti-Myc antibody (left panel). Each Western blot was probed with an anti-GAPDH antibody to control for loaded protein (right panel). The figure shows one representative blot out of at least three independent experiments.

FIGURE 4.

Pharmacological interaction between first and second generation GSMs. Graphical analysis of modulator cross-competition in a cellular assay for Aβ42. A–F, graphs display AZ4800 versus the following: A, AZ4800; B, AZ3303; C, AZ1136; D, E2012; E, (R)-flurbiprofen; and F, sulindac sulfide. Parallel shift of the lines (A–D) indicates competitive binding to the same or overlapping sites, and intercepting lines (E and F) suggest binding noncompetitive with AZ4800. Lines are fitted to the data by linear regression (mean ± S.E., n = 2). v is amount Aβ42 generated/min analyzed in conditioned media from HEK/APPswe cells.

FIGURE 5.

Binding distribution of ³H-labeled GSM and GSI to rodent brain sections and comparison with APP immunohistochemistry. 5 nm ³H-labeled GSM AZ8349 (A) and 5 nm ³H-labeled GSI DBZ (C) display excellent anatomical binding overlap to sagittal brain sections from guinea pig. Note very high binding associated to the subventricular zone (SVZ). Nonspecific binding (5 nm ³H-labeled GSM AZ8349 + 5 μm unlabeled AZ8349) is shown (B). D–G, images of immunohistochemistry detecting APP in coronal brain sections from TG2576 mice (E–G) and WT controls (D and E). Note the stronger overall APP-like immunoreactivity in TG2576 mice compared with WT. In contrast, ³H-labeled GSM AZ8349 has no increased binding in TG2576 (I) compared with WT (H). Quantification of binding in the autoradiograms as optical density (photostimulated luminescence (PSL)/mm²) is shown in the bar graphs.

FIGURE 6.

Displacement of ³H-labeled GSM by different GSMs on rat cryo-cut brain sections. A, rat slices (coronal, 10 μm) were either incubated with 5 nm [³H]AZ8349 alone (panel i) together with 0.5 mm (R)-flurbiprofen (panel ii) or together with 1 μm AZ4800 (panel iii). AZ4800, but not (R)-flurbiprofen, could displace the specific binding of [³H]AZ8349. B, graphical display illustrating the displacement binding studies of 5 nm [³H]AZ8349. Both AZ GSMs (10 μm) and E2012 displace [³H]AZ8349, whereas neither sulindac sulfide 0.1 mm nor (R)-flurbiprofen does (0.5 mm), indicating distinct interaction points. Binding is quantified as PSL/mm², and data are presented as means ± S.E., n = 3. Tot, total; PSL, photostimulated luminescence.

FIGURE 7.

Effect of AZ GSMs on brain Aβ42 levels in C57BL/6 mice. Acute per oral dosing of AZ3303 (top) (100 and 300 μmol/kg) and AZ4800 (bottom) (75, 150, and 300 μmol/kg) causes a statistically significant decrease of Aβ42 levels in diethylamine-extracted brain homogenates at 1.5 h post-drug administration compared with the vehicle group in 12–18-week-old female C57BL/6 mice (n = 8–9 mice/group). Statistical analysis: one-way analysis of variance followed by Dunnett's multiple comparison test (**, p < 0.01; *, p < 0.05). LOQ, limit of quantitation.