Article Figures & Data
Figures
- Figure 1.
In vitro pharmacological profiling of MRK-560 and other GSIs. A, MRK-560 inhibition of Aβ peptide generation and NICD translocation in HEK293 cells stably expressing APPswe and ΔENotch, respectively. B, MRK-560 inhibition of Aβ42 and Aβ40 generation in HEK_PS1 and HEK_PS2 cells transiently transfected with C99 (black, Aβ42; gray, Aβ40). C, MRK-560 inhibition of Aβ42 generation in BD8_PS1 and BD8_PS2 cells transiently transfected with C99. D, MRK-560 inhibition of NICD and AICD in HEK_PS1 and HEK_PS2 cells (black, NICD; gray, AICD). E, DBZ inhibition of Aβ42 peptide generation and NICD translocation in HEK293 cells stably expressing APPswe and ΔENotch and Aβ42 generation in BD8_PS1 and BD8_PS2 cells transiently transfected with C99 (squares, Aβ42(BD8)). F, Semagecestat inhibition of Aβ42 generation and NICD translocation in HEK293 cells stably expressing APPswe and ΔENotch, respectively. G, Avagecestat inhibition of Aβ42 generation and NICD translocation in HEK293 cells stably expressing APPswe and ΔENotch, respectively (black, Aβ42; gray, NICD in E–G). The effect on Aβ42 production and NICD and AICD formation was analyzed after 5, 12, and 16 h, respectively, and plotted (mean ± SEM, n = 3, n = 2 for NICD and AICD, and n = 1 for Aβx-37, 38, and 39) relative to controls (0.5% DMSO and 0.5 μm L685458).
- Figure 2.
MRK-560 displays differential binding to PS1 and PS2. A, B, Displacement binding studies of [3H]DBZ to membranes from HEK_PS1 and HEK_PS2 cells, respectively. DBZ (A) showed equipotent and full inhibition of [3H]DBZ binding to both membrane preparations whereas MRK-560 (B) was more potent in displacing [3H]DBZ from HEK_PS1 compared with HEK_PS2 membranes, but achieved only partial displacement of [3H]DBZ. C–E, Displacement binding studies of [3H]DBZ to brain sections from PS2-deficient mice. C, Total binding of 2 nM [3H]DBZ. D, Complete displacement of 2 nM [3H]DBZ by 1 mM DBZ. E, Partial displacement of 2 nM [3H]DBZ by 1 mM MRK-560.
- Figure 3.
MRK-560 reduces Aβ40 and Aβ42 levels in plasma and brain. The levels of Aβ40 and Aβ42 were measured 3 h after the final administration of MRK-560 (7.5 and 30 μmol/kg) to WT (A, C), PS2-deficient mice (B, D), and Tg2576 mice (E–H). Data are presented as mean ± SEM with lower limit of quantification (LOQ) illustrated.
- Figure 4.
MRK-560 induces Notch-related histopathology in PS2-deficient mice. Histological sections from jejunum (A–C), thymus (D–F), and spleen (G–I) from WT (A, B, D, E, G, H) and PS2-deficient (PS2−/−) (C, F, I) mice. The mice were treated with MRK-560 at 30 μmol/kg (B, C, E, F, H, I) or vehicle (A, D, G). PS2-deficient mice treated with MRK-560 showed goblet cell metaplasia (arrows) in the jejunum (C), atrophy of the thymic cortex (Cx) (F), and a reduction of the marginal zone (Mz) in the spleen (I). WT mice treated with MRK-560 (B, E, H) did not differ from vehicle-treated controls (A, D, G). H&E staining. Magnifications: 10× (A–C, G–I), 4× (D–F), and inset in the lower left corner 40×. J–O, Grading of Notch-related histopathology in mice treated with MRK-560 (J–L) and DBZ (M–O). The histopathological score (0–5) is shown on the y-axis, and the treatment group on the x-axis. p values refer to Dunn's multiple-comparison test. J, M, Thymus atrophy. K, N, Goblet cell metaplasia. L, O, Lymphoid depletion (marginal zone) in the spleen.
- Figure 5.
γ-Secretase expression in brain, thymus, and spleen of WT and PS2-deficient mice. A–C, Western blot analysis of membrane extracts from brain (A), spleen (B), and thymus (C) from WT (+) and PS-deficient (−) mice revealed that PS1 and PS2 are expressed in all tissues examined (with the exception that PS2 is not expressed in PS2-deficient mice), and that PS1 is more highly expressed than PS2 in all three tissues. PS1 NTF, Presenilin 1 N-terminal fragment; PS2 CTF, Presenilin 2 C-terminal fragment; Pen-2, Presenilin enhancer 2. D–F, Specific binding of 0.5 nM [3H]DBZ in crude plasma membrane fraction from brain (D), spleen (E), and thymus (F) showed that binding was not significantly different when WT (+) was compared with PS2-deficient (−) tissue. Nonspecific binding was defined in the presence of 1 μm semagecestat. G, RT-PCR analysis of PS2 and β-actin (housekeeping gene as control) mRNA levels in jejunum from C57BL/6 mice exposed to vehicle (V) or MRK-560 (7.5 μmol/kg and 30 μmol/kg, respectively) for 4 d. Each group represents six mice and the mean values of PS2 expression levels normalized to the β-actin expression levels are drawn in the graph.
Tables
- Table 1.
Exposure levels of MRK-560 and DBZ in plasma and brain of wild-type (A), PS2-deficient (B), and Tg2576 (C) mice 3 h after final dose on the fourth day
Compound (Route) Dose (μmol/kg) Cp (nm) Cbr, corr (nm) Cu,p (nm) Cu,br, corr (nm) A MRK-560 (s.c.) 7.5 3013 656 12 4 MRK-560 (s.c.) 30 10843 2081 43 14 DBZ (s.c.) 100 109 55 — — B MRK-560 (s.c.) 7.5 3980 666 16 4 MRK-560 (s.c.) 30 14690 3638 59 24 DBZ (s.c.) 100 770 9291 — — C MRK-560 (p.o.) 7.5 537 134 4 1 MRK-560 (p.o.) 30 1494 531 6 4 Cp, Plasma concentration; Cbr, corr, brain concentration corrected for plasma; Cu, p, free plasma concentration; Cu, br, corr, free brain concentration corrected for plasma.
