Semagacestat (LY450139) (LY450139) is a γ-secretase blocker for Aβ42/Aβ40/Aβ38 (IC50: 10.9/12.1/12.0 nM) and also inhibits Notch signaling (IC50: 14.1 nM).

γ-secretase (Aβ42):10.9 nM, γ-secretase (Aβ38):12.0 nM, γ-secretase (Aβ40):12.1 nM

Semagacestat通过降低从稳定过表达人类野生型APP的H4人类胶质瘤细胞中向培养基中分泌的Aβ42、Aβ40和Aβ38，其IC50分别为10.9 nM、12.1 nM和12.0 nM，同时不影响细胞活性。此外，Semagacestat能够增加细胞裂解液中的β-CTF，其ECmax为16.0 nM，且在高浓度时该增加效应会出人意料地减弱。Semagacestat抑制Notch信号传导，IC50为14.1 nM，对Notch的选择性极小，Notch IC50/Aβ42 IC50仅为1.3。[1] 对于表达内源性小鼠APP的小鼠CTX，Semagacestat导致Aβ40在介质中浓度依赖性下降，IC50为111 nM，但小鼠Aβ42的形成量大约是Aβ40的12倍，这与野生型小鼠神经元的数据相符。[2]

经口给予5.5个月大的APP转基因Tg2576小鼠Semagacestat（1 mg/kg），能显著改善使用Y迷宫任务测量的空间工作记忆缺陷，但在连续服用8天后，该效果消失。LY450139在剂量为10 mg/kg时能使海马Aβ42和Aβ40水平分别降低22-23%，在30 mg/kg时降低36-41%，并在0.3-10 mg/kg剂量依赖性增加β-CTF，但不抑制大脑中其他γ-分泌酶底物（如Notch, N-钙黏蛋白或EphA4）的处理。然而，LY450139在野生型小鼠和3月龄Tg2576小鼠中损害了正常的认知功能，未能在Y迷宫测试中恢复认知缺陷。[1]

Cellular APP processing assay and Notch signaling assay: H4 human glioma cells stably overexpressing human wild-type APP695 are treated with Semagacestat at various concentrations for 24 hours. Levels of Aβ42, Aβ40, and Aβ38 in the media are measured using separate ELISA kits. The expression vector of the constitutively active form of Notch (NotchΔE), encoding bases 1-60 and 5193-6657 of the human Notch1 coding region (NM_017617), is constructed into a pcDNA3.1 vector with a sequence modification from mouse to human. Notch signaling activity is evaluated using Cignal RBP-Jk Reporter Assay kit. RBP-Jk protein [CSL/CBF1/Su(H)/Lag1] is a transcription factor activated with Notch intracellular domain produced by γ-secretase. H4 cells are transiently transfected with the human NotchΔE expression vector and the RBP-Jk-responsive luciferase construct using Lipofectamine 2000, and then exposed to various concentrations of Semagacestat for 16 hours. Notch signaling is measured based on luciferase activity in the cell lysate using the Dual-Glo Luciferase Assay System.

Cells are incubated with Semagacestat for 24 hours. For detection of cell viability, the percentage of viable cells is quantified by their capacity to reduce 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) following incubation with 0.5 mg/mL MTT for 60 minutes. For the detection of sAPP species, cells are lysed and analysed by western blotting.(Only for Reference)