Piperidine acetic acid based γ-secretase modulators directly bind to Presenilin-1

Abstract

Aβ42 is believed to play a causative role in Alzheimer's disease (AD) pathogenesis. γ-Secretase modulators (GSMs) are actively being pursued as potential AD therapeutics because they selectively alter the cleavage site of the amyloid precursor protein (APP) to reduce the formation of Aβ42. However, the binding partner of acid based GSMs was unresolved until now. We have developed clickable photoaffinity probes based on piperidine acetic acid GSM-1 and identified PS1 as the target within the γ-secretase complex. Furthermore, we provide evidence that allosteric interaction of GSMs with PS1 results in a conformational change in the active site of the γ-secretase complex leading to the observed modulation of γ-secretase activity.

Figure 1

Structure of piperidine acetic acid GSMs 1 and 2 and clickable photoaffinity probe analogues 3–5 designed to label target proteins upon UV-irradiation. The labeled proteins are then reacted with an azide-linked reporter tag for further characterization.

Figure 2

(a) Illustration of the preparation of PS1ΔE9 proteoliposomes for γ-secretase activity assay or photolabeling. (b) PS1ΔE9 proteoliposomes were labeled with 4 or 5 in the presence or absence of 10 μM 1, followed by click chemistry with TAMRA-azide, in-gel fluorescence, and Coomassie blue staining.

Figure 3

(a) HeLa membranes were labeled with 4 or 5 (top panel) or labeled with 5 in the presence or absence of 50 μM 1 or 2 (bottom panel), followed by click chemistry with biotin-azide, streptavidin pull down, and Western blot analysis with PS1-NTF antibody. (b) HeLa membranes were labeled with 150 nM 5 in the presence or absence of 10 μM 1, followed by click chemistry with TAMRA-azide, in gel fluorescence, and Coomassie blue gel staining.

Figure 4

(a) HeLa membranes were labeled with 20 nM of GSI photoprobe L646, GY4, JC8, or L505 in the presence or absence of 4 μM 1, followed by streptavidin pull down and Western blot analysis with PS1-NTF antibody. (b) Densitometry analysis from PS1-NTF labeling of GY4 with coincubation of 1 at 4 or 12 μM. (c) Proposed model for the mechanism of action of acid GSMs. GSM binding to PS1 allosterically influences the S1 subsite within the active site, leading to an alteration of γ-secretase specificity and an observed increase in GY4 labeling. R = biotin linker.