Clickable gamma‐secretase modulator photoaffinity probes bind distinct sites on presenilin

Abstract

γ‐Secretase is an intramembrane aspartyl protease that cleaves the amyloid precursor protein to generate Aβ species, including the neurotoxic Aβ‐42. Aβ‐42 is a component of β‐amyloid plaques, and is believed to play a causative role in Alzheimer's disease (AD). γ‐Secretase modulators (GSMs) have emerged as a potential treatment for AD because they decrease the production of Aβ‐42 without affecting the processing of other critical γ‐secretase substrates. We used clickable GSM photoaffinity probes to identify the protein target of different classes of GSMs and to probe their mechanism‐of‐action. Probes (i.e., E2012‐BPyne and GSM‐5) were synthesized with a benzophenone or aryl azide for photocrosslinking the compound to the target proteins and an alkyne handle for click chemistry. Cell membranes or live cells were treated with the GSM probe with or without competitor compounds from various GSM and γ‐secretase inhibitor (GSI) classes. Samples were UV‐irradiated followed by click chemistry with biotin‐azide, enriched on Streptavidin and detected by Western blotting. These studies revealed distinct allosteric binding sites on the N‐terminal fragment of presenilin 1 (PS1‐NTF). Furthermore, E2012‐BPyne labeling of PS1‐NTF was potentiated by the presence of the active site‐directed GSI, L458, suggesting a degree of cooperativity between the protein's active site and modulatory binding sites of certain GSMs.