Development of a high-throughput assay for screening of gamma-secretase inhibitor with endogenous human, mouse or Drosophila gamma-secretase

Abstract

Selective lowering of amyloid-beta levels with small-molecule gamma-secretase inhibitors is a promising therapeutic approach for Alzheimer's disease. In this work, we developed a high throughput assay for screening of gamma-secretase inhibitors with endogenous gamma-secretase and a fluorogenic substrate. The IC(50) values of known gamma-secretase inhibitors generated with this method were comparable with reported values obtained by other methods. The assay was optimized and applied to a small-scale screening of 1,280 compounds. The discovery of several new inhibitors warrants further investigation. This assay was also proven to be easily adopted to test compounds for drosophila and mouse gamma-secretase, which could be very useful to assess compounds activity against gamma-secretase from different species before the in vivo test in animal models.

Figure 1

Assay optimization. A. Western blot analysis of the expression of PS-1 in HEK293T cells. HEK293T cells were transfected with a plasmid encoding the N-terminal fragment of human PS1 (PS1-NTF) or not (HEK293T), and the samples were subjected to western blot assay. B. The sequence of APP with the γ-secretase cleavage sites. C. The sequence of the fluorogenic substrate. D. Various amount of membrane proteins were incubated with 4, 6 or 8 μM of fluorogenic substrate for 5 h with or without the presence of 1 μM L-685,458. The γ-secretase activity was presented as the relative fluorescent unit (RFU). E. The specific γ-secrease activity increases with the increase of substrate concentration. F. 10 μg of membrane proteins were incubated with 6 μM of substrate for various periods of time, and γ-secretase activity were measured. Data was shown as Mean±SEM of at least three independent experiments. (n ≥ 3).

Figure 2

Assay Performance. (A) Z’ factor determination. At the optimized conditions (10 μg membrane proteins, 6 μM substrate, 5 h incubation time), 48 replicates of positive (wells treated with 1% DMSO) and negative signals (wells treated with 1 μM L685,458) were studied. Dashed lines indicate means ± 3 SD of 48 data points. (B) Reproducibility. The corresponding wells from two different 96-well plates were inhibited with same concentration of L685,458. The reproducibility of data from duplicate plates was investigated with linear repression analysis.

Figure 3

Dose-response curves of known inhibitors against human γ-secretase. Six γ-secretase inhibitors were tested on human γ-secretase obtained from HEK293T cell membrane with the fluorogenic substrate assay. Data are mean ± SEM (n ≥ 3).

Figure 4

Dose-response curves of known inhibitors against drosophila and mouse γ-secretase. Four γ-secretase inhibitors were tested on drosophila γ-secretase obtained from S2 cell membrane (A) and mouse γ-secretase from mouse cortex membrane with the fluorogenic substrate assay. Data are mean ± SEM (n = 3).

Figure 5

High-throughput screening of 1280 compounds. (A) Result of the primary screening of 1280 compounds in duplicate setup with HEK293T membrane. Compounds with more than 60% inhibition of the γ-secretase were further tested. (B) In the secondary screening with a triplet setup, seven compounds displayed consistant inhibitory effect compare to the known γ-secretase inhibitor L685,458.